Methods for inducing cell division of postmitotic cells

ABSTRACT

The present disclosure provides methods for inducing cell cycle reentry of postmitotic cell. The present disclosure further provides cells and compositions for treating diseases, such as cardiovascular diseases, neural disorders, hearing loss, and diabetes.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a 371 U.S. National Stage Application ofInternational Patent Application No. PCT/US2016/026059 filed Apr. 5,2016, which claims priority from U.S. Provisional Application62/144,244, filed Apr. 7, 2015 and U.S. Provisional Application62/151,321, filed Apr. 22, 2015 which are incorporated herein byreference in their entireties.

BACKGROUND

Regenerative medicine is the replacement, engineering and regenerationof cells, tissues and organs to gain or restore normal cellularfunction. Much of the regenerative medicine field is concentrated oncellular replacement therapies, particularly stem cell-based therapies.One property of stem cells that makes them uniquely suited forcell-based applications is their ability to proliferate in culture forprolonged periods of time. This proliferative capacity offers a sourceof starting material from which to derive sufficient numbers of cellsfor use in cell-based applications. However, as cells age, even manystem cells grown in culture, their proliferative capacity tends todecrease.

In addition, many adult cells, including cardiomyocytes, neurons, andskeletal muscle, are considered to be postmitotic cells that achievegrowth through hypertrophy rather than hyperplasia. Postmitotic cellsare unable, or have an extremely limited ability, to divide orregenerate. As such, many organs containing these postmitotic cells areseverely restricted in their ability to adequately repair or restorefunction after any significant injury.

It would be beneficial to provide methods for reinitiating postmitoticcell division to regenerate cells and/or replace damaged cells. Suchcapabilities would be useful for both in vitro culturing and screeningas well as in vivo applications for the treatment of numerous diseasesand disorders.

SUMMARY

This disclosure is predicated on the discovery that certain agents caninduce proliferation and/or cell cycle reentry of postmitotic cells andis directed, in part, to methods of inducing proliferation and/or cellcycle reentry of a postmitotic cell, comprising contacting thepostmitotic cell with an effective amount of a composition comprising atleast one cyclin-dependent kinase (CDK) and at least one cyclin, orequivalents of each thereof, thereby inducing proliferation and/or cellcycle reentry of the postmitotic cell.

One aspect of the disclosure provides an isolated, proliferativepostmitotic cell modified to overexpress at least one CDK and at leastone cyclin, or an equivalent of each thereof. Another aspect of thedisclosure provides an isolated, proliferative postmitotic cell modifiedto overexpress CDK1 and CCNB1, or an equivalent of each thereof.

One aspect of the disclosure provides a method for treating acardiovascular disease comprising administering to a subject in needthereof, an effective amount of a composition that increases theexpression of CDK1 and CCNB1, or an equivalent of each thereof.

Another aspect of the disclosure provides a method for treating aneurological disease comprising administering to a subject in needthereof an effective amount of a composition that increases theexpression of CDK1 and CCNB1, or equivalents thereof.

In some embodiments, the methods further comprise contacting the cellwith an effective amount of a CDK activator, a transforming growthfactor β inhibitor, or combinations thereof. In some embodiments, theCDK activator is a CDK1 activator.

In some embodiments, the contacting is conducted in vitro or in vivo.

In some embodiments, the postmitotic cell is selected from the groupconsisting of a cardiomyocyte, a neural cell, a pancreatic cell, a haircell, and a skeletal muscle cell. In one preferred embodiment, theproliferative postmitotic cells are proliferative cardiomyocytes. Insome embodiments, the proliferative cardiomyocytes are modified tooverexpress CDK1 and CCNB1, or equivalents thereof. In otherembodiments, the proliferative cardiomyocytes are modified tooverexpress CDK4, CCND1, or both. In another preferred embodiment, theproliferative postmitotic cells are proliferative neural cells. In someembodiments, the proliferative neural cells are modified to overexpressCDK1 and CCNB1, or equivalents of each thereof. In other embodiments,the proliferative neural cells are modified to overexpress CDK4, CCND1,or both.

In some embodiments, the composition comprises at least one nucleicacid. In some embodiments, the cyclin-dependent kinase (CDK) and/or thecyclin is encoded by a nucleic acid. In some embodiments, the at leastone nucleic acid is a modified mRNA. In other embodiments, the at leastone nucleic acid is constitutively expressed.

In some embodiments, the cyclin is selected from the group consisting ofcyclinA, cyclinB, cyclinD, and cyclinE. In some embodiments, the cyclinBis cyclinB1 (CCNB1). In some embodiments, the cyclinD is cyclinD1(CCND1).

In some embodiments, the CDK is selected from the group consisting ofCDK1, CDK2, CDK3, CDK4, and CDK6. In one preferred embodiment, the CDKis CDK1. In another preferred embodiment, the CDK is CDK4. In someembodiments, wherein the CDK is constitutively expressed.

In some embodiments, the CDK is CDK1 and the cyclin is CCNB1.

In some embodiments, the composition further comprises CDK4, CCND1, orboth.

In some embodiments, the cells are further modified to overexpress CDK4,CCND1, or both.

In some embodiments, the methods comprise administering an effectiveamount of a composition that increases the expression of CDK4, CCND1, orboth.

Another aspect provides a method for treating a cardiovascular diseasecomprising administering to a subject in need thereof, an effectiveamount of a composition that increases the expression of CDK1 and CCNB1,or an equivalent of each thereof.

A further aspect provides a method for treating a cardiovascular diseasecomprising administering to a subject in need thereof, an effectiveamount of a population of the proliferative postmitotic cells disclosedand described herein.

A further aspect provides a method for treating a neurological diseasecomprising administering to a subject in need thereof, an effectiveamount of a population of the proliferative postmitotic cells disclosedand described herein.

In yet a further aspect is provided a method for treating a neurologicaldisease comprising administering to a subject in need thereof aneffective amount of a composition that increases the expression of CDK1and CCNB1, or equivalents thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts selected microarray data showing differentially expressedgenes related to cell cycle regulation between neonatal (newborn P0) andadult (6 week-old) mouse cardiac cells. Data are represented as log₂,comparing adult and newborn cardiac cells.

FIG. 2 demonstrates that overexpression of several of the topdifferentially regulated genes between proliferative (neonatal) andnon-proliferative (adult) cardiac cells promote cardiac cellproliferation in mouse cardiomyocytes (panel A). Cardiomyocytestransduced with a control, CDK1, CCNB1, or AURKB expressing adenoviruswere stained with EDU proliferation marker, Troponin T cardiac markerand DAPI nuclear stain. All three genes enhanced cardiac proliferation(panel B).

FIG. 3 demonstrates that overexpression of CDK1, CCNB1 and AURKB promotecardiac cell proliferation in human induced pluripotent stem cell(hiPSC)-derived cardiomyocytes. Human iPSC-derived cardiomyocytesoverexpressing CDK1, CCNB1 and AURKB show increased expression of theproliferation marker phospho histone H3 (PHH3).

FIG. 4 shows increase in cardiomyocytes number following overexpressionof CDK1, CCNB1 and AURKB for 72 hours in hiPSC-derived cardiomyocytesusing three different techniques to quantify cardiomyocyte cell number.Panel A shows cell doubling in the total number of nuclei in response tooverexpression of CDK1, CCNB1 and AURKB and approximately 30% of totalnuclei stained positive for PHH3. Fluorescence activated cell sorting(FACS) also revealed an increase in total cell number followingoverexpression of CDK1, CCNB1, and AURKB in hiPSC-derived cardiomyocytes(panel B). An ATP cellular content analysis showed a two-fold increasein cell number following overexpression of CDK1, CCNB1 and AURKB inhiPSC-derived cardiomyocytes (n=3, *p<0.05) (panel C).

FIG. 5 depicts time lapse imaging of cell division in hiPSC-derivedcardiomyocytes overexpressing CDK1, CCNB1 and AURKB. Panels 1-15 arerepresentative images collected every hour for four days. Panel 16 showsthat cells at the end of the imaging period stain positive for cardiacmarker Troponin T. Arrows denote two dividing cells and their progeny.

FIG. 6 depicts time lapse imaging of cell division in adult mousecardiomyocytes isolated from α-MHC-GFP mice overexpressingconstitutively active CDK1 (CDK1AF), CCNB1 and AURKB. Panels 1-8 arerepresentative images collected every hour for four days showing celldivision of a cardiomyocyte.

FIG. 7 demonstrates that overexpression of constitutively active CDK1(CDK1AF), CCNB1 and AURKB promote cardiac cell proliferation in mouseprimary cardiomyocytes. Images are representative images of adult mousecardiomyocytes transduced with a control vector (panel A) or CDK1AF,CCNB1 and AURKB (panel B) and stained with phospho histone H3 (PHH3).Arrows denote dividing cells.

FIG. 8 shows results from a proof of principle study in mice. CDK1AF,CCNB1 and AURKB or GFP adenovirus were injected in c57bl6/N hearts atthe site of injury following myocardial infarction (MI). Hearts wereeither harvested five days post infarct (panel A) to evaluate cellproliferation (PHH3 staining) or six weeks post infarct (panel B) toevaluate cardiac function and histology. Hearts harvested at six weeksfollowing MI were fixed and stained for fibrosis using Masson Trichromestain.

FIG. 9 shows results from the cardiac function assessment for the proofof principle study in mice. CDK1AF, CCNB1 and AURKB or GFP controladenovirus were injected in c57bl6/N hearts at the site of injuryfollowing myocardial infarction (MI). The animals were followed byechocardiography to assess the heart function 3-, 10-, 24- and 40-dayspost MI by measuring % ejection fraction (panel A) and decline inejection fraction over time (panel B).

FIG. 10 shows the ability of various combinations of factors to inducecell cycle reentry of human cardiomyocytes as assessed by cellproliferation (PHH3 staining) (panel A) and survival by total nucleicounts (panel B). Cells were assessed at 4-, 8-, and 12-days followingtreatment.

FIG. 11 demonstrates the differences in cell cycle distribution betweena first cocktail of CDK1, CCNB, and Aurora (panel A), a cocktail of CDK4and CCND (panel B), and a cocktail of CDK1, CDK4, CCNB and CCND (panelC).

FIG. 12 shows improved cardiac function following injection of CDK1AF,CCNB, CDK4, CCND (G1/G2 cocktail) adenoviruses into the myocardium atthe time of myocardial infarction as indicated by the ejection fraction(EF) measured by echocardiography.

FIG. 13 depicts histological sections taken from control and treatedanimals showing thicker muscles (panel A) and reduced scar size (panelB) in treated animals.

FIG. 14 demonstrates dividing cardiomyocytes treated with the CDK1AF,CCNB, CDK4, CCND cocktail using mosaic analysis of dual marker (MADM)anaylsis.

FIG. 15 show time lapse images of a neuron making three successive celldivisions within five days when treated with the CDK1AF, CCNB, CDK4,CCND cocktail.

FIG. 16 demonstrates that cells treated with the CDK1AF, CCNB, CDK4,CCND cocktail undergo most divisions 60-90 hours post-infection, withsome cell division occurring as late as 120 hours post-infection. Thecolumn height represents the number of cells that have undergonedivision and the number on the top of the column indicates the averagenumber of divisions performed by each cell.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of this disclosure will be limited only by theappended claims.

The detailed description of the disclosure is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. Although any methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated by reference to disclose and describe the methods and/ormaterials in connection with which the publications are cited.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, cell biology and recombinant DNA, which are withinthe skill of the art. See, e.g., Sambrook and Russell eds. (2001)Molecular Cloning: A Laboratory Manual, 3^(rd) edition; the seriesAusubel et al. eds. (2007) Current Protocols in Molecular Biology; theseries Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson etal. (1991) PCR 1: A Practical Approach (IRL Press at Oxford UniversityPress); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow andLane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)Culture of Animal Cells: A Manual of Basic Technique, 5^(th) edition;Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson(1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984)Transcription and Translation; IRL Press (1986) Immobilized Cells andEnzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Millerand Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (ColdSpring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) GeneSilencing by RNA Interference: Technology and Application (CRC Press);Sell (2013) Stem Cells Handbook.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 1.0, where appropriate. It isto be understood, although not always explicitly stated that allnumerical designations are preceded by the term “about.” It also is tobe understood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acardiomyocyte” includes a plurality of cardiomyocytes.

I. Definitions

As used herein the following terms have the following meanings.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5% or1%.

“Administration,” “administering” and the like, when used in connectionwith a composition of the disclosure refer both to directadministration, which may be administration to cardiac cells in vitro,administration to cardiac cells in vivo, administration to a subject bya medical professional or by self-administration by the subject and/orto indirect administration, which may be the act of prescribing acomposition of the disclosure. When used herein in reference to a cell,refers to introducing a composition to the cell. Typically, an effectiveamount is administered, which amount can be determined by one of skillin the art. Any method of administration may be used. For example, if acomposition of nucleic acids or polypeptides are being administered to apostmitotic cell, the skilled artisan may use transduction,transfection, or the like to administer the nucleic acids orpolypeptides to the cell. In addition, small molecules may beadministered to the cells by, for example, addition of the smallmolecules to the cell culture media or injection in vivo to site ofcardiac injury. “Administration, “administering” and the like as usedherein in reference to a subject in need thereof refers to introducing acomposition of proliferation and/or cell cycle reentry factors orproliferative postmitotic cells into a subject in need thereof.Administration to a subject can be achieved by, for example,intravascular injection, intramyocardial delivery, intracranialdelivery, and the like.

As used herein, the term “cell cycle reentry” refers to the process of apostmitotic cardiac cell reinitiating at least one stage of the cellcycle, for example, DNA synthesis, mitosis, karyokinesis andcytokinesis.

As used herein the term “cardiac cell” refers to any cell present in theheart that provides a cardiac function, such as heart contraction orblood supply, or otherwise serves to maintain the structure of theheart. Cardiac cells as used herein encompass cells that exist in theepicardium, myocardium or endocardium of the heart. Cardiac cells alsoinclude, for example, cardiac muscle cells or cardiomyocytes, and cellsof the cardiac vasculatures, such as cells of a coronary artery or vein.Other non-limiting examples of cardiac cells include epithelial cells,endothelial cells, fibroblasts, cardiac stem or progenitor cells,cardiac conducting cells and cardiac pacemaking cells that constitutethe cardiac muscle, blood vessels and cardiac cell supporting structure.Cardiac cells may be derived from stem cells, including, for example,embryonic stem cells or induced pluripotent stem cells. The cells can beof any appropriate species, e.g., an animal such as a mammal, e.g., acanine, an equine, a feline, or a human cell.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

As used herein, the term “derived” means differentiated from,reprogrammed from, isolated from or otherwise purified. For example, acardiomyocyte derived from a fibroblast is a cardiomyocyte that has beenreprogrammed in vitro or in vivo from a fibroblast, for example, byoverexpressing the GMT cocktail of factors.

As used herein the term “effective amount” and the like in reference toan amount of a composition of proliferation and/or cell cycle reentryfactors refers to an amount that is sufficient to induce proliferationand/or cell cycle reentry of cardiac cells (e.g., cardiomyocytes). Thecells are contacted with amounts of the composition of proliferationand/or cell cycle reentry factors effective to induce cell cycle reentryand/or proliferation. When used herein in reference to administration toa subject in need thereof, the terms “effective amount” mean an amountof a composition of proliferation and/or cell cycle reentry factors orpostmitotic cells induced to proliferate and/or reenter the cell cyclewhich treat a disease. An effective amount can be administered in one ormore administrations, applications or dosages. Such delivery isdependent on a number of variables including the time period which theindividual dosage unit is to be used, the bioavailability of thecomposition, the route of administration, etc. It is understood,however, that specific amounts of the compositions (e.g., ofproliferation and/or cell cycle reentry factors or proliferative cardiaccells) for any particular subject depends upon a variety of factorsincluding the activity of the specific agent employed, the age, bodyweight, general health, sex, and diet of the subject, the time ofadministration, the rate of excretion, the composition combination,severity of the particular disease being treated and form ofadministration.

As used herein, the term “equivalents thereof” refers to a polypeptideor nucleic acid sequence that differs from a reference polypeptide ornucleic acid sequence (i.e., a cyclin protein or fragment thereofconsistent with embodiments of the present disclosure), but retainsessential properties (i.e., biological activity). A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, deletions, additions, fusions and truncations in thepolypeptide encoded by the reference sequence. Generally, differencesare limited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical.

As used herein, “induce,” “inducing” and the like when used in referenceto proliferation and/or cell cycle reentry means that postmitotic cellsreplicate at a faster rate and/or more frequently. In some embodimentsof this and other aspects described herein, postmitotic cellproliferation is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%,70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, 100-fold or more higher relative to anuntreated control. The % or fold increase in postmitotic cellproliferation can be determined by measuring the number of replicatingpostmitotic cells following administration of a composition capable ofstimulating proliferation and/or cell cycle reentry, as describedherein, relative to a control where the postmitotic cells are not incontact with the composition. Increase in proliferation can also bebased on ratios of replicating cells to total number of cells in therespective treated and untreated control. In some embodiments, totalnumber of cells in the treated and untreated controls is used todetermine the proliferation. Cell proliferation can be determined usingthe BrdU incorporation method (and similar methods, for example, EdUincorporation) described in Yamagishi et al. (2001) Dev Biol 239:190-203, the content of which is incorporated herein by reference. Othermethods of detecting cell proliferation include use of antibodies toKi67 for G1, S, G2 and M-phase and phospho histone 3 (PHH3) for M-phaseof the cell cycle.

As used herein the term “isolated” with reference to a cell, refers to acell that is in an environment different from that in which the cellnaturally occurs, e.g., where the cell naturally occurs in amulticellular organism, and the cell is removed from the multicellularorganism, the cell is “isolated.” An isolated modified postmitotic cellcan be present in a mixed population of genetically modified postmitoticcells, or in a mixed population comprising genetically modifiedpostmitotic cells and postmitotic cells that are not geneticallymodified. For example, an isolated genetically modified postmitotic cellcan be present in a mixed population of genetically modified postmitoticcells in vitro, or in a mixed in vitro population comprising geneticallymodified postmitotic cells and postmitotic cells that are notgenetically modified. The term “isolated” as used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs or RNAs, respectively that are present in the natural sourceof the macromolecule. The term “isolated nucleic acid” is meant toinclude nucleic acid fragments which are not naturally occurring asfragments and would not be found in the natural state. The term“isolated” is also used herein to refer to polypeptides, proteins thatare isolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides. In other embodiments, the term“isolated” means separated from constituents, cellular and otherwise, inwhich the cell, tissue, polynucleotide, peptide, polypeptide, protein,antibody or fragment(s) thereof, which are normally associated innature. For example, an isolated cell is a cell that is separated formtissue or cells of dissimilar phenotype or genotype. As is apparent tothose of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, does not require “isolation” todistinguish it from its naturally occurring counterpart.

As used herein, the term “nucleic acid” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include linear andcircular nucleic acids, messenger RNA (mRNA), cDNA, recombinantpolynucleotides, vectors, probes, and primers.

The terms “polypeptide,” “peptide,” and “protein,” are usedinterchangeably herein and refer to a polymeric form of amino acids ofany length, which can include genetically coded and non-geneticallycoded amino acids, chemically or biochemically modified or derivatizedamino acids, and polypeptides having modified peptide backbones. Theterm includes fusion proteins, including, but not limited to, fusionproteins with a heterologous amino acid sequence, fusions withheterologous and homologous leader sequences, with or without N-terminalmethionine residues, immunologically tagged proteins, and the like.

The term “proliferation” as used herein refers to growth and division ofpostmitotic cells. In some embodiments, the term “proliferation” is usedherein in reference to postmitotic cells refers to a group ofpostmitotic cells that increase in number over a period of time. Theterm “proliferation and/or cell cycle reentry factors” refers to anyagent that can induce, alone or in combination with additional agents, apostmitotic cell to undergo or enter DNA synthesis, mitosis,karyokinesis and/or cytokinesis.

As used herein, the term “progenitor cell” refers to a cell that iscommitted to differentiate into a specific type of cell or to form aspecific type of tissue. A progenitor cell, like a stem cell, canfurther differentiate into one or more kinds of cells, hut is moremature than a stem cell such that it has a more limited/restricteddifferentiation capacity.

As used herein the term “subject” refers to a mammal, preferably ahuman, but includes and is not limited to non-human primates, murines(i.e., mice and rats), canines, felines, equines, bovines, ovines,porcines, caprines, etc. In some embodiments, the subject is a humansubject.

“Treatment,” “treating,” and “treat” are defined as acting upon adisease, disorder, or condition with an agent to reduce or ameliorateharmful or any other undesired effects of the disease, disorder, orcondition and/or its symptoms. “Treatment,” as used herein, covers thetreatment of a subject in need thereof, and includes treatment ofcardiovascular disease (e.g., heart failure, myocardial ischemia,hypoxia, stroke, myocardial infarction and chronic ischemic heartdisease), a neurological disease (e.g., epilepsy, Alzheimer disease andother dementias, cerebrovascular diseases including stroke, migraine andother headache disorders, multiple sclerosis, Parkinson's disease,neuroinfections, brain tumors, traumatic disorders), diabetes, orhearing impairment or loss. “Treating” or “treatment of” a condition orsubject in need thereof refers to (1) taking steps to obtain beneficialor desired results, including clinical results such as the reduction ofsymptoms; (2) preventing the disease, for example, causing the clinicalsymptoms of the disease not to develop in a patient that may bepredisposed to the disease but does not yet experience or displaysymptoms of the disease; (3) inhibiting the disease, for example,arresting or reducing the development of the disease or its clinicalsymptoms; (4) relieving the disease, for example, causing regression ofthe disease or its clinical symptoms; or (5) delaying the disease. Forone purpose of this invention, beneficial or desired clinical resultsinclude, but are not limited to, inducing proliferation of a cardiaccell, inducing cell cycle reentry, and/or promoting myocardialregeneration. For another purpose of this invention, beneficial ordesired clinical results include, but are not limited to, inducingproliferation of a neuron, inducing cell cycle reentry, and/or promotingneuronal regeneration.

The term “vector” is used herein to refer to a nucleic acid moleculecapable of transferring or transporting another nucleic acid molecule.The transferred nucleic acid is generally linked to, for example, thevector nucleic acid molecule. A vector may include sequences that directautonomous replication in a cell, or may include sequences sufficient toallow integration into cardiac cell DNA. Useful vectors include, forexample, plasmids (e.g., DNA plasmids or RNA plasmids), transposons,cosmids, bacterial or yeast artificial chromosomes and viral vectors.Useful viral vectors include, for example, adenoviruses, retroviruses,particularly replication defective retroviruses, and lentiviruses.

As used herein, the term “viral vector” refers either to a nucleic acidmolecule that includes virus-derived nucleic acid elements thattypically facilitate transfer of the nucleic acid molecule orintegration into the genome of a cell or to a viral particle thatmediates nucleic acid transfer. Viral particles will typically includevarious viral components and sometimes also cell components in additionto nucleic acid(s). The term “viral vector” may also refer either to avirus or viral particle capable of transferring a nucleic acid into acell or to the transferred nucleic acid itself. Viral vectors andtransfer plasmids contain structural and/or function genetic elementsthat are primarily derived from a virus. The viral vector may be ahybrid vector, LTR or other nucleic acid containing both retroviral(e.g., lentiviral) sequences and non-retroviral viral sequences. Ahybrid vector may refer to a vector or transfer plasmid comprisingretroviral (e.g., lentiviral) sequences for reverse transcription,replication, integration and/or packaging.

The term “adenoviral vector” as used herein, refers to any adenoviralvector that includes exogenous DNA which encodes a polypeptide insertedinto its genome. The vector must be capable of replicating and beingpackaged when any deficient essential genes are provided in trans. Anadenoviral vector desirably contains at least a portion of each terminalrepeat required to support the replication of the viral DNA, preferablyat least about 90% of the full ITR sequence, and the DNA required toencapsidate the genome into a viral capsid. Many suitable adenoviralvectors have been described in the art. U.S. Pat. No. 6,440,944; seeU.S. Pat. No. 6,040,174 (replication defective E1 deleted vectors andspecialized packaging cell lines). In some embodiments, the adenoviralexpression vector is one that is replication defective in normal cells.In other embodiments, an adenoviral vector refers to an adeno-associatedviral (AVV) vector. In some embodiments, the adenoviral expressionvector is pseudotyped to enhance targeting.

The term “retroviral vector” refers to a viral vector or plasmidcontaining structural and functional genetic elements, or portionsthereof, that are primarily derived from a retrovirus.

The term “lentiviral vector” refers to a viral vector or plasmidcontaining structural and functional genetic elements, or portionsthereof, that are primarily derived from a lentivirus.

The terms “lentiviral vector” or “lentiviral expression vector” may beused to refer to lentiviral transfer plasmids and/or infectiouslentiviral particles. It is understood that nucleic acid sequenceelements such as cloning sites, promoters, regulatory elements,heterologous nucleic acids, etc. are present in RNA form in thelentiviral particles of the invention and are present in DNA form in theDNA plasmids of the invention.

The term “genetic modification” refers to a permanent or transientgenetic change induced in a cell following introduction of new nucleicacid (i.e., nucleic acid exogenous to the cell). Genetic change can beaccomplished by incorporation of the new nucleic acid into the genome ofthe cardiac cell, or by transient or stable maintenance of the newnucleic acid as an extrachromosomal element. Where the cell is aeukaryotic cell, a permanent genetic change can be achieved byintroduction of the nucleic acid into the genome of the cell. Suitablemethods of genetic modification include viral infection, transfection,conjugation, protoplast fusion, electroporation, particle guntechnology, calcium phosphate precipitation, direct microinjection, andthe like.

The terms “regenerate,” “regeneration” and the like as used herein inthe context of injured cells or tissue shall be given their ordinarymeanings and shall also refer to the process of growing and/ordeveloping new cells or tissue (e.g., cardiac) in tissue that has beeninjured, for example, injured due to ischemia, infarction, reperfusion,or other disease. In some embodiments, tissue regeneration comprisesactivation and/or enhancement of cell proliferation. In someembodiments, tissue regeneration comprises activation and/or enhancementof cell migration.

The term “stem cells” refer to cells that have the capacity toself-renew and to generate differentiated progeny. The term “pluripotentstem cells” refers to stem cells that can give rise to cells of allthree germ layers (endoderm, mesoderm and ectoderm), but do not have thecapacity to give rise to a complete organism. In some embodiments, thecompositions for inducing proliferation and/or cell cycle reentry can beused on a population of stem cells to increase proliferation rates. Inother embodiments, the compositions do not increase proliferation ratesof stem cells.

The term “induced pluripotent stem cells” shall be given its ordinarymeaning and shall also refer to differentiated mammalian somatic cells(e.g., adult somatic cells, such as skin) that have been reprogrammed toexhibit at least one characteristic of pluripotency. See, for example,Takahashi et al. (2007) Cell 131(5):861-872, Kim et al. (2011) Proc.Natl. Acad. Sci. 108(19): 7838-7843, Sell (2013) Stem Cells Handbook.

The term “postmitotic cell” is a cell that does not exhibit mitosis orcellular division. Non-limiting examples of postmitotic cells includedifferentiated cells that comprise the brain (e.g., neurons), heart(e.g., cardiomyocytes) and skeletal muscle. As used herein, the term“proliferative postmitotic cell” refers to a postmitotic cell that hasbeen induced to proliferate and/or reenter the cell cycle. For example,a proliferative cardiomyocyte is a cardiomyocyte, having none or limitedproliferative capacity, that has been induced to proliferate and/orreenter the cell cycle. A proliferative postmitotic cell (e.g.,proliferative cardiomyocyte) may undergo any number of cell divisions,for example, one, two, three, four, five, six, seven, eight, nine, ten,fifteen, twenty, or more cell divisions before again exiting the cellcycle and returning to a postmitotic state.

II. Methods of Inducing Proliferation and/or Cell Cycle Reentry ofPostmitotic Cells

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure provides methods of inducing proliferation and/orcell cycle reentry of a postmitotic cell, the methods comprising, oralternatively consisting essentially of, or yet further consisting of,contacting the postmitotic cell with an effective amount of acomposition in an amount effective to stimulate proliferation and/orcell cycle reentry of the postmitotic cell (e.g., one or moreproliferation and/or cell cycle reentry factors). The cell can be fromany species, an animal, a mammal, e.g., a canine, a feline, a murine, arat, or a human cell.

In some embodiments, the contacting is conducted in vitro or in vivo.

In one aspect this disclosure provides, methods of inducingproliferation and/or cell cycle reentry of a postmitotic cell (e.g., apostmitotic cardiomyocyte), the method comprising, or alternativelyconsisting essentially of, or yet further consisting of, contacting thepostmitotic cell with an effective amount of a composition to stimulateproliferation and/or cell cycle reentry of the postmitotic cell. Thecomposition is capable of increasing expression of at least one geneencoding a cyclin-dependent kinase (CDK), a cyclin, an aurora kinase,actin binding protein anillin (ANLN), a cell division cycle (CDC)protein, a cadherin, a COPS signalosome complex subunit, a cullin, aGTPase-activating protein, a protein regulator of cytokinesis and/orWNT1-inducible signaling pathway protein or equivalents thereof. Thecell can be from any species, an animal, a mammal, e.g., a canine, afeline, a murine, a rat, or a human cell.

In some embodiments, the postmitotic cell is selected from the groupconsisting of a cardiomyocyte, a neural cell, a pancreatic cell, a haircell, and a skeletal muscle cell.

Cardiac Cells

The cardiac cells of the present disclosure include any cells present inthe heart that provide a cardiac function. Cardiac cells encompass cellsthat exist in the epicardium, myocardium or endocardium of the heart.Cardiac cells also include, for example, cardiac muscle cells orcardiomyocytes, and cells of the cardiac vasculatures, such as cells ofa coronary artery or vein. Other non-limiting examples of cardiac cellsinclude epithelial cells, endothelial cells, fibroblasts, cardiac stemor progenitor cells, cardiac conducting cells and cardiac pacemakingcells that constitute the cardiac muscle, blood vessels and cardiac cellsupporting structure.

Cardiac cells can be derived from cardiac or non-cardiac cells. Cardiaccells can be from or derived from any of a variety of tissue sources.For example, stem cells, cardiac fibroblasts, foreskin fibroblast,dermal fibroblasts, lung fibroblasts, etc. The cardiac cells can beembryonic, fetal, or post-natal (e.g., adult) cardiac cells. In apreferred embodiment, the cardiac cells are adult cardiac cells. In someembodiments, the cardiac cells are derived from stem cells.

Non-cardiac cells can be differentiated into cardiac cells in vitro orin vivo using any method available to one of skill in the art. Forexample, see methods described in leda M. et al. (2010) Cell142(3):375-386 and Kwon C. et al. Proc. Natl. Acad. Sci. (2007)104(26):10894-10899.

In certain embodiments, the cardiac cells are cardiomyocytes. In certainembodiments, the cardiomyocytes are adult postmitotic cardiomyocytes. Inanother aspect, the adult postmitotic cardiomyocytes have no or lowproliferative capacity. In some embodiments, the cardiomyocyte is amononucleated cell. In other embodiments, the cardiomyocyte ismulti-nucleated.

Where the cells for modification are a population of cardiac cells, thepopulation of cells is composed of at least about 60% cardiac cells, atleast about 65% cardiac cells, at least about 70% cardiac cells, atleast about 75% cardiac cells, at least about 80% cardiac cells, atleast about 85% cardiac cells, at least about 90% cardiac cells, atleast about 95% cardiac cells, at least about 98% cardiac cells, atleast about 99% cardiac cells, or greater than 99% cardiac cells.

Neural Cells

The neural cells of the present disclosure include any cells present inthe nervous system that provide a neural function. Neural cellsencompass cells that exist in the brain (e.g., cerebrum, cerebellum, andbrainstem) and spinal cord, as well as cells of the peripheral nervoussystem, including sensory neurons. Neural cells also include, forexample, neurons, oligodendrocytes, and astrocytes. Non-limitingexamples of neurons include, motor neurons, pyramidal neuron, purkinjecells, retinal neuron, olfactory neuron, touch and pain sensory neuron,and amacrine cells.

Neural cells can be derived from neural or non-neural cells. Neuralcells can be from or derived from any of a variety of tissue sources.For example, stem cells, fibroblasts, foreskin fibroblast, dermalfibroblasts, lung fibroblasts, etc. The neural cells can be embryonic,fetal, or post-natal (e.g., adult) neural cells. In a preferredembodiment, the neural cells are adult neural cells. In someembodiments, the neural cells are derived from stem cells (e.g., neuralstem cells).

Non-neural cells can be differentiated into neural cells in vitro or invivo using any method available to one of skill in the art. For example,see methods described in Guo et al. (2014) Cell Stem Cell 14(2):188-202;Keirstead et al. (2005) J Neurosci. 25(19):4694-4705; Kim et al. (2012)Curr Opinion Neurobiol. 22(5):778-784.

In certain embodiments, the neural cells are neurons. In certainembodiments, the neurons are adult postmitotic neurons. In preferredembodiments, the adult postmitotic neurons have no or low proliferativecapacity.

Where the cells for modification are a population of neural cells, thepopulation of cells is composed of at least about 60% neural cells, atleast about 65% neural cells, at least about 70% neural cells, at leastabout 75% neural cells, at least about 80% neural cells, at least about85% neural cells, at least about 90% neural cells, at least about 95%neural cells, at least about 98% neural cells, at least about 99% neuralcells, or greater than 99% neural cells.

Pancreatic Cells

The pancreatic cells of the present disclosure include any cells presentin the pancreas that provide a pancreatic function. Examples ofpancreatic cells include cells within the islets of Langerhans (e.g.,alpha cells, beta cells, delta cells, PP cells and epsilon cells) andacinar cells. In some embodiments, the pancreatic cells are pancreaticprogenitor cells.

Pancreatic cells can be derived from pancreatic or non-pancreatic cells.Pancreatic cells can be from or derived from any of a variety of tissuesources. For example, stem cells, fibroblasts, foreskin fibroblast,dermal fibroblasts, lung fibroblasts, etc. The pancreatic cells can beembryonic, fetal, or post-natal (e.g., adult) pancreatic cells. In apreferred embodiment, the pancreatic cells are adult pancreatic cells.In some embodiments, the pancreatic cells are derived from stem cells.

Non-pancreatic cells can be differentiated into pancreatic cells invitro or in vivo using any method available to one of skill in the art.For example, see methods described in D'Amour et al. (2005) NatureBiotech. 23:1534-1541 and U.S. Pat. No. 8,633,024.

In certain embodiments, the pancreatic cells are beta cells or beta cellprecursors. In certain embodiments, the beta cells are adult beta cells.In preferred embodiments, the adult postmitotic pancreatic cells have noor low proliferative capacity.

Where the cells for modification are a population of pancreatic cells,the population of cells is composed of at least about 60% pancreaticcells, at least about 65% pancreatic cells, at least about 70%pancreatic cells, at least about 75% pancreatic cells, at least about80% pancreatic cells, at least about 85% pancreatic cells, at leastabout 90% pancreatic cells, at least about 95% pancreatic cells, atleast about 98% pancreatic cells, at least about 99% pancreatic cells,or greater than 99% pancreatic cells.

Hair Cells

Hair cells of the present disclosure include inner hair cells(stereocilia) and outer hair cells. Hair cells can be derived from hairor non-hair cells. Hair cells can be from or derived from any of avariety of tissue sources. For example, stem cells, fibroblasts,foreskin fibroblast, dermal fibroblasts, lung fibroblasts, etc. The haircells can be embryonic, fetal, or post-natal (e.g., adult) hair cells.In a preferred embodiment, the hair cells are adult hair cells. In someembodiments, the hair cells are derived from stem cells. Non-hair cellscan be differentiated into hair cells in vitro or in vivo using anymethod available to one of skill in the art. For example, see methodsdescribed in Mohammad et al. (2014) Stem Cells and Dev.23(11):1275-1284. In preferred embodiments, the adult postmitotic haircells have no or low proliferative capacity.

Where the cells for modification are a population of hair cells, thepopulation of cells is composed of at least about 60% hair cells, atleast about 65% hair cells, at least about 70% hair cells, at leastabout 75% hair cells, at least about 80% hair cells, at least about 85%hair cells, at least about 90% hair cells, at least about 95% haircells, at least about 98% hair cells, at least about 99% hair cells, orgreater than 99% hair cells.

Skeletal Muscle Cells

Skeletal muscle cells of the present disclosure refer to cells of themuscle fibers. Skeletal muscle cells can be derived from skeletal musclecells or non-skeletal muscle cells. Skeletal muscle cells can be from orderived from any of a variety of tissue sources. For example, stemcells, fibroblasts, foreskin fibroblast, dermal fibroblasts, lungfibroblasts, etc. The skeletal muscle cells can be embryonic, fetal, orpost-natal (e.g., adult) skeletal muscle cells. In a preferredembodiment, the skeletal muscle cells are adult skeletal muscle cells.In some embodiments, the skeletal muscle cells are derived from stemcells. Non-skeletal muscle cells can be differentiated into muscle cellsin vitro or in vivo using any method available to one of skill in theart. For example, see methods described in Salani et al. (2012) J CellMol. Med. 16(7):1353-1364. In preferred embodiments, the adultpostmitotic skeletal muscle cells have no or low proliferative capacity.

Where the cells for modification are a population of skeletal musclecells, the population of cells is composed of at least about 60%skeletal muscle cells, at least about 65% skeletal muscle cells, atleast about 70% skeletal muscle cells, at least about 75% skeletalmuscle cells, at least about 80% skeletal muscle cells, at least about85% skeletal muscle cells, at least about 90% skeletal muscle cells, atleast about 95% skeletal muscle cells, at least about 98% skeletalmuscle cells, at least about 99% skeletal muscle cells, or greater than99% skeletal muscle cells.

In some embodiments the postmitotic cells are endogenous postmitoticcells such that the cells are within the subject and the methods ofinducing proliferation and/or cell cycle reentry are by in vivomodification. In other embodiments, the postmitotic cells are exogenousand the postmitotic cells are modified in vitro.

The postmitotic cells that are induced to proliferate and/or reenter thecell cycle can be from any of a variety of sources. Mammalianpostmitotic cells (e.g., human, canine, feline or murine) can be used.In some embodiments, the postmitotic cells are mammalian cardiomyocytes.In some embodiments, the postmitotic cells can be derived from stemcells (e.g., pluripotent stem cells, induced pluripotent stem cells,reprogrammed cardiac cells, cardiac stem cells, neural stem cells). Insome embodiments, embryonic stem cells are expressly excluded.

The postmitotic cells can be obtained from a living subject. The cellscan be obtained from tissue taken from a living subject. The cells canbe obtained from a recently deceased subject who is considered asuitable tissue donor. In some embodiments, the subject is screened forvarious genetic disorders, viral infections, etc. to determine whetherthe subject is a suitable source of cells. In general, a cell that issuitable for use in the present invention is non-transformed (e.g.,exhibits normal cell proliferation) and is otherwise normal (e.g.,exhibits normal karyotype).

Postmitotic cells can be derived from tissue of a non-embryonic subject,a neonatal infant, a child or an adult. Postmitotic cells can be derivedfrom neonatal or post-natal tissue collected from a subject within theperiod from birth, including cesarean birth, to death. For example, thepostmitotic cell induced to proliferate and/or reenter the cell cyclecan be from a subject who is greater than about 10 minutes old, greaterthan about 1 hour old, greater than about 1 day old, greater than about1 month old, greater than about 2 months old, greater than about 6months old, greater than about 1 year old, greater than about 2 yearsold, greater than about 5 years old, greater than about 10 years old,greater than about 15 years old, greater than about 18 years old,greater than about 25 years old, greater than about 35 years old, >45years old, >55 years old, >65 years old, >80 years old, <80 years old,<70 years old, <60 years old, <50 years old, <40 years old, <30 yearsold, <20 years old or <10 years old.

Methods of isolating postmitotic cells from tissues are known in theart, and any known method can be used. As a non-limiting example, adultcardiac cells can be obtained from human heart atrial biopsy specimensobtained from patients undergoing cardiac surgery. Cardiac tissue can beminced and digested with collagenase and cardiac stem/progenitor cellsexpanded in c-kit+ progenitor cell expansion media using the methods ofChoi et al. (2013) Transplantation Proceedings 45:420-426. In addition,cardiac fibroblasts can be obtained using the methods of leda et al.(2009) Dev. Cell 16(2):233-244. Foreskin fibroblasts can be obtainedfrom foreskin tissue of a male individual. The fibroblasts can beobtained by mincing the foreskin tissue, then dissociating the tissue tosingle cells. Foreskin cell clumps can be dissociated by any means knownin the art including physical de-clumping or enzymatic digestion using,for example trypsin.

Postmitotic cells (e.g., cardiomyocytes, neural cells, pancreatic cells,hair cells, skeletal muscle cells) can be genetically or non-geneticallymodified with one or more nucleic acids comprising nucleotide sequencesencoding cell cycle regulating genes, for example, a cyclin-dependentkinase, a cyclin and/or an aurora kinase, or can be modified byintroducing polypeptides. As discussed below, a postmitotic cell can beinduced to proliferate or reenter the cell cycle by overexpressingnucleic acids comprising nucleotide sequences encoding acyclin-dependent kinase, a cyclin and/or an aurora kinase (e.g., CDK1,CCNB1 and AURKB), or by introducing polypeptides comprising acyclin-dependent kinase, a cyclin and/or an aurora kinase amino acidsequences (e.g., CDK1, CCNB1 and AURKB), or still further modified byintroducing chemicals (e.g., small molecules) to induce expression ofendogenous a cyclin-dependent kinase, a cyclin, and/or an aurora kinase(e.g., CDK1, CCNB1 and AURKB). Amino acid sequences for cyclin-dependentkinases, cyclins and aurora kinases are known in the art. Nucleotidesequences encoding cyclin-dependent kinases, cyclins and aurora kinasesare known in the art.

Additionally, a postmitotic cell can be induced to proliferate orreenter the cell cycle by overexpressing nucleic acids comprisingnucleotide sequences encoding at least one cyclin-dependent kinase (CDK)and at least one cyclin, or equivalents of each thereof (e.g., CDK1 andCCNB1) or by introducing polypeptides comprising at least onecyclin-dependent kinase and at least one cyclin amino acid sequences(e.g., CDK1 and CCNB1), or still further modified by introducingchemicals (e.g., small molecules) to induce expression of endogenous acyclin-dependent kinase and/or a cyclin.

In some embodiments, the postmitotic cell is contacted with an effectiveamount of a CDK activator, a transforming growth factor β inhibitor, orcombinations thereof. In some embodiments, the CDK activator is a CDK1activator. An example of a CDK1 activator is the Weel inhibitor MK1775.MK1775 is known to indirectly induce CDK1 expression. A transforminggrowth factor beta (TGF-β) inhibitor is a compound that inhibits TGF-βsignal transduction by inhibiting any of the factors constituting theTGF-β signal transduction system pathway, such as TGF-β ligand, TGF-βType I receptors, TGF-β Type II receptors, TGF-β Type III receptors(β-glycan and endoglin), soluble forms of the TGF-β receptors, Smadproteins, antibodies against receptors and ligands implicated in thesignaling pathway, nucleic acid based molecules (e.g., antisense, siRNA,aptamers and ribozymes) targeting the pathway members, or a combinationthereof.

In some embodiments, the TGF-β inhibitor is selected from the groupconsisting of SB431542, D4476, LDN-193189, dexamethasone and LY364947.TGF-β inhibitors also may be referred to in the art as anti-TGF-βcompounds. Non-limiting examples of anti-TGF-β compounds include,antibodies (e.g., Fresolumimab/GC1008 (Genzyme, Cambridge, Mass., USA),PF-03446962 (Pfizer, New York, N.Y., USA)), antisense oligonucleotides(ASO) (e.g., Trabedersen (AP12009) (Isarna Therapeutics, New York, N.Y.,USA)), receptor kinase inhibitors (e.g., LY2157299 (Eli Lilly,Indianapolis, Ind., USA), and combined TGF-β ASO with a vaccine (e.g.,Lucanix™ (Belagenpumatucel-L) (Nova Rx Corp, San Diego, Calif., USA),and TGF-β₂ ASO+GMCSF expression vector (Mary Crowley Medical ResearchCentre, Dallas, Tex., USA)). It is contemplated that addition of a TGF-βinhibitor to the composition acts to improve cell survival.

Culture Conditions

The cells of the present disclosure can be cultured under any conditionsknown to one of skill in the art. In some embodiments, the cells arecultured in conditions of 1-20% oxygen (O₂) and 5% carbon dioxide (CO₂).In some embodiments, the cells of the present disclosure are culturedunder hypoxic conditions (e.g., in the presence of less than 10% O₂). Insome embodiments, the cells of the present disclosure are cultured atabout 37° C. In some embodiments, the cells of the present disclosurecan be cultured at about 37° C., 5% CO₂ and 10-20% O₂. In someembodiments, the cells are cultured in hypoxic conditions for a periodof time. For example, the cells may be cultured under normoxicconditions (˜20% O₂) for a period of time and then switched to hypoxicconditions, for example ˜5% O₂.

In Vitro Modification

In one aspect, the methods and compositions of the present disclosureinduce proliferation and/or cell cycle reentry of a postmitotic cell, bycontacting the postmitotic cell with an effective amount of acomposition comprising at least one cyclin-dependent kinase (CDK) and atleast one cyclin, or equivalents of each thereof, thereby inducingproliferation and/or cell cycle reentry of the postmitotic cell.

In one aspect, the methods and compositions of the present disclosureinduce postmitotic cell proliferation and/or cell cycle reentry byadministering to a postmitotic cell a composition capable of stimulatingproliferation and/or cell cycle reentry (e.g., a composition capable ofincreasing expression of a cyclin-dependent kinase, a cyclin, and/or anaurora kinase).

In some embodiments, the compositions induce the postmitotic cells todivide in vitro over a period of at least one day, of at least two days,of at least three days, of at least four days, of at least five days, ofat least six days, of at least seven days, of at least eight days, or ofat least nine days in vitro. In one embodiment, at least about 0.1% ofthe postmitotic cells are induced to proliferate and/or reenter the cellcycle. In another embodiment at least about 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 10%, 15% of the postmitotic cellsare induced to proliferate and/or reenter the cell cycle.

In some embodiments, a composition to induce postmitotic cellproliferation and/or cell cycle reentry is administered to thepostmitotic cell in vitro or ex vivo. The postmitotic cells are modified(genetically or non-genetically) to stimulate proliferation and/or cellcycle reentry in vitro or ex vivo. Once at least a portion of thepostmitotic cells have at least begun to proliferate and or reenter thecell cycle in vitro or ex vivo, the postmitotic cells can be introducedinto a subject. In some embodiments, the postmitotic cells of thepresent disclosure can be used as a utility in research and drugdevelopment. Postmitotic cells may be cultured by a variety of methodscommonly known to those of skill in the art.

The advantage of in vitro or ex vivo modification of postmitotic cellsis the ability to easily identify cells suitable for implantation or fordiscrimination of cells that are damaged or are not proliferating and/orhave not reentered the cell cycle. In vitro or ex vivo modificationallows postmitotic cells that have been modified to be purified orisolated from those postmitotic cells that have not been modified.

Postmitotic cells may be modified by a variety of mechanisms commonlyknown to those of skill in the art. Viral constructs can be deliveredthrough the production of a virus in a suitable host. Virus is thenharvested from the host cell and contacted with the postmitotic cell.Viral and non-viral vectors capable of expressing genes of interest canbe delivered to a postmitotic cell via DNA/liposome complexes, micellesand targeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. In addition to the delivery of polynucleotides to apostmitotic cell or cell population, direct introduction of proteinsdescribed herein to the postmitotic cell or cell population can be doneby the non-limiting technique of protein transfection, alternativelyculturing conditions that can enhance expression and/or promote activityof the proteins of this invention are other non-limiting techniques.

Other methods of delivering vectors encoding genes of the currentinvention include, but are not limited to, calcium phosphatetransfection, DEAE-dextran transfection, electroporation,microinjection, protoplast fusion, or liposome-mediated transfection.The host cells that are transfected with the vectors of this disclosuremay include, but are not limited to, E. coli or other bacteria, yeast,fungi, or cells derived from mice, humans, or other animals (e.g.,mammals). In vitro expression of a protein, fusion, polypeptide fragmentor mutant encoded by cloned DNA may also be used. Those skilled in theart of molecular biology will understand that a wide variety ofexpression systems and purification systems may be used to producerecombinant proteins and fragments thereof.

In Vivo Modification

In some embodiments, the present disclosure provides methods of inducingpostmitotic cell proliferation and/or cell cycle reentry in vivo. Insome embodiments, the compositions induce the postmitotic cells todivide in vivo over a period of at least one day, of at least two days,of at least three days, of at least four days, of at least five days, ofat least six days, of at least seven days, of at least eight days, or ofat least nine days in vivo. In one embodiment, at least about 0.1% ofthe postmitotic cells are induced to proliferate and/or reenter the cellcycle. In another embodiment at least about 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 10%, 15% of the postmitotic cellsare induced to proliferate and/or reenter the cell cycle.

In some embodiments, a postmitotic cell is modified (e.g., geneticallyor non-genetically) in vivo with an effective amount of a composition tostimulate proliferation and/or cell cycle reentry of the postmitoticcell (e.g., cardiomyocytes), wherein the composition is capable ofincreasing expression of a cyclin-dependent kinase, a cyclin, and anaurora kinase, or equivalents thereof and wherein the composition isadministered to a subject in vivo. In other embodiments, a postmitoticcell is modified (e.g., genetically or non-genetically) in vivo with aneffective amount of a composition comprising at least onecyclin-dependent kinase (CDK) and at least one cyclin, or equivalents ofeach thereof, thereby inducing proliferation and/or cell cycle reentryof the postmitotic cell in vivo. The compositions described herein cancontain nucleic acids, polypeptides, and/or combinations thereof.

For in vivo delivery of a composition to stimulate proliferation and/orcell cycle reentry of a postmitotic cell (e.g., postmitoticcardiomyocytes) the composition can be administered to a subject in needthereof by any mechanisms commonly known to those of skill in the art.Non-limiting examples include oral, systemic (e.g., transdermal,intranasal or by suppository), or parenteral (e.g., intramuscular,intravenous or subcutaneous) administration.

The present disclosure provides methods of stimulating proliferationand/or cell cycle reentry of a postmitotic cell in vivo, the methodgenerally comprising, or alternatively consisting essentially of, or yetfurther consisting of administering to a subject in need thereof anamount effective to stimulate proliferation and/or cell cycle reentry,wherein said composition is capable of increasing expression of acyclin-dependent kinase, a cyclin, and an aurora kinase, or equivalentsthereof. In some embodiments, the methods generally involve contacting apostmitotic cell (e.g., a postmitotic cardiomyocyte) with a compositionin an amount effective to stimulate proliferation and/or cell cyclereentry. The composition can comprise at least one proliferation and/orcell cycle reentry factor comprising, or alternatively consistingessentially or, or yet further consisting of, at least one polypeptide,at least one nucleic acid, at least one chemical, or a mixture thereofto increase expression of a cyclin-dependent kinase, a cyclin, and/or anaurora kinase, or equivalents thereof. The composition can comprises, oralternatively consists essentially or, or yet further consists of, atleast one proliferation and/or cell cycle reentry factor comprising, atleast one polypeptide, at least one nucleic acid, at least one chemical,or a mixture thereof to increase expression of at least onecyclin-dependent kinase and at least one cyclin, or equivalents thereof.In some embodiments the composition comprises, or alternatively consistsessentially or, or yet further consists of, a CDK1, CCNB1 and an AURKBpolypeptide. In some embodiments the composition comprises, oralternatively consists essentially or, or yet further consists of, aCDK1 and a CCNB1 polypeptide. In some embodiments the compositioncomprises, or alternatively consists essentially or, or yet furtherconsists of, a CDK1, CCNB1, CDK4, and CCND1 polypeptide. In anotherembodiment, the composition comprises, or alternatively consistsessentially or, or yet further consists of, a CDK1, CCNB1 and an AURKBnucleic acid. In another embodiment, the composition comprises, oralternatively consists essentially or, or yet further consists of, aCDK1 and CCNB1 nucleic acid. In another embodiment, the compositioncomprises, or alternatively consists essentially or, or yet furtherconsists of, a CDK1, CCNB1, CDK4, and CCND1 nucleic acid. In otherembodiments, the composition comprises, or alternatively consistsessentially or, or yet further consists of, a chemical that increasesexpression of endogenous CDK1, CCNB1 and AURKB. In other embodiments,the composition comprises, or alternatively consists essentially or, oryet further consists of, a chemical that increases expression ofendogenous CDK1 and CCNB1. In yet other embodiments, the compositioncomprises, or alternatively consists essentially or, or yet furtherconsists of, a chemical that increases expression of endogenous CDK1,CCNB1, CDK4, and CCND1. In some embodiments, an aurora kinase isexpressly excluded from the composition.

The composition can be a solid composition, a semi-solid composition, ora liquid composition. In some cases the composition is a controlledrelease composition, which may be a solid composition, a semi-solidcomposition, or a liquid composition. For example, the composition canbe a sustained release matrix.

In some embodiments, the composition is administered to a subject inneed thereof at or near a treatment site, for example, in or around theheart or brain. Administration of the composition can be achieved byvarious means, including via intravascular injection, intramyocardialdelivery, and intracranial delivery. For example, intramyocardialdelivery can be carried out using a catheter (e.g., via atransendocardial catheter system). Intramyocardial or intracranialdelivery via a catheter can be global, focal or diffuse.

In some embodiments, the postmitotic cells which have been induced toproliferate or reenter the cell cycle are administered into a subject inneed thereof in association with an implantable device. Suitableimplantable devices contemplated by this invention include intravascularstents (e.g., self-expandable stents, balloon-expandable stents, andstent-grafts), scaffolds, grafts, and the like.

Factors to Stimulate Proliferation and/or Cell Cycle Reentry

As discussed, a postmitotic cell (e.g., a cardiomyocyte) may be modifiedto increase expression of a cell cycle regulating gene. The cell cycleregulating gene, for example, a cyclin-dependent kinase (CDK), cyclin,and/or aurora kinase can be encoded by a nucleic acid or can be apolypeptide. In some embodiments, the cell cycle regulating geneincludes a cyclin-dependent kinase, a cyclin, an aurora kinase, orequivalents thereof. In other embodiments, the cell cycle regulatinggene includes a cyclin-dependent kinase and a cyclin or an equivalent ofeach thereof. A postmitotic cell can be modified by introduction of acomposition comprising proliferation and/or cell cycle reentry factors,such as at least one of a cyclin-dependent kinase, a cyclin, an aurorakinase, actin binding protein anillin (ANLN), a cell division cycle(CDC) protein, a cadherin, a COPS signalosome complex subunit, a cullin,a GTPase-activating protein, a protein regulator of cytokinesis and/orWNT1-inducible signaling pathway protein or equivalents thereof. Apostmitotic cell can be modified by introduction of a compositioncomprising proliferation and/or cell cycle reentry factors such as: (1)cyclinB1 (CCNB1), aurora kinase B (AURKB) and cycle dependent kinase 1(CDK1); (2) CCNB1 and CDK1; (3) cyclinD1 (CCND1), cycle dependent kinase4 (CDK4); (4) CCNB1, CDK1, CCND1 and CDK4; (5) CCNB1, CCND1 and CDK4; orany combination thereof.

In some embodiments, the at least one nucleic acid is constitutivelyexpressed.

Cyclin-Dependent Kinases

Cyclin-dependent kinases (CDKs) function in regulating progressionthrough the cell cycle by complexing with their regulatory cyclins. Forexample, CDK1 binds cyclinB1 (CCNB1) and functions during M phase, whilethe CDK2/cyclinA complex ensures progression in S phase and S/G2transition and CDK2/cyclinE promotes progression during G1/S phase.Gerard et al. (2012) Frontiers in Physiol. 3(413):1-18.

In some embodiments, the CDK is selected from the group consisting ofCDK1, CDK2, CDK3, CDK4, and CDK6. In some embodiments, the CDK is CDK1.In other embodiments, the CDK is CDK4. In yet other embodiments, bothCDK1 and CDK4 are used. In some embodiments, the CDK is constitutivelyexpressed.

Amino acid sequences for CDKs (e.g., CDK1) and nucleotide sequencesencoding CDK polypeptides, from a variety of species, are known in theart. See, e.g.: (1) GenBank Accession No. NP_001777.1 (Homo sapiens 297amino acid cyclin-dependent kinase 1 isoform 1); (2) GenBank AccessionNo. AAH14563.1 (Homo sapiens 297 amino acid cell division cycle 2, G1 toS and G2 to M); (3) GenBank Accession No. NM_001786.4 (nucleotidesequence encoding the Homo sapiens cyclin-dependent kinase 1 (CDK1),transcript variant 1); (4) GenBank Accession No. NM_033379.4 (nucleotidesequence encoding the Homo sapiens cyclin-dependent kinase 1 (CDK1),transcript variant 1); (5) GenBank Accession No. NP_031685.2 (Musmusculus 297 cyclin-dependent kinase 1); (6) GenBank Accession No.NM_007659.3 (nucleotide sequence encoding the Mus musculuscyclin-dependent kinase 1).

In some embodiments, a suitable CDK1 nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, or 100% nucleotide sequenceidentity of SEQ ID NO. 1 or SEQ ID NO. 7.

In some embodiments, a suitable CDK1 nucleic acid comprises a nucleotidesequence encoding a CDK1 polypeptide, where in some embodiments, asuitable CDK1 polypeptide comprises an amino acid sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99%, or 100% amino acid sequence identity of SEQ IDNO. 2 or SEQ ID NO. 8.

In some embodiments, a suitable CDK4 nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, or 100% nucleotide sequenceidentity of SEQ ID NO. 13 or SEQ ID NO. 17.

In some embodiments, a suitable CDK4 nucleic acid comprises a nucleotidesequence encoding a CDK4 polypeptide, where in some embodiments, asuitable CDK4 polypeptide comprises an amino acid sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99%, or 100% amino acid sequence identity of SEQ IDNO. 14 or SEQ ID NO. 18.

Cyclins

A cyclin functions to control the progression of cells through the cellcycle by activating cyclin-dependent kinases (CDK). Galderisi et al.(2003) Oncogene 22(33): 5208-5219. Cyclin-CDK complexes activate otherproteins through phosphorylation, which in turn are responsible forspecific events in the cell cycle. Cyclins are well-characterized anddivided into families. For example, the cyclinA family consists of twomembers, CCNA1 and CCNA2, while the cyclinB family consists of threemembers, CCNB1, CCNB2 and CCNB3. Different cyclins are active duringdifferent phases of the cell cycle. For example, cyclinA is active inthe synthesis phase (S phase) during which DNA is replicated and occursbetween G1 phase and G2 phase. On the other hand, CyclinD regulatestransition from G1 to S phase. CyclinB regulates progression from G2 toM phase. In particular, CyclinB1 functions as a mitotic cyclin thatbinds to CDK1 and is necessary for the progression of cells in and outof M phase of the cell cycle.

In some embodiments, the cyclin is selected from the group consisting ofcyclinA, cyclinB, cyclinD, and cyclinE. In some embodiments, the cyclinBis cyclinB1 (CCNB1). In other embodiments, the cyclinD is cyclinD1(CCND1). In yet other embodiments, both CCNB1 and CCND1 are used.

Amino acid sequences for cyclin polypeptides (e.g., cyclinB1, CCNB1) andnucleotide sequences encoding cyclin polypeptides (e.g., cyclinB1,CCNB1), from a variety of species, are known in the art. See, e.g.: (1)GenBank Accession No. NP_114172 (Homo sapiens 433 amino acid Cyclin B1);(2) GenBank Accession No. AAH06510.1 (Homo sapiens 433 amino acid CyclinB1); (3) GenBank Accession No. EAW51306.1 (Homo sapiens 433 amino acidCyclin B1); (4) GenBank Accession No. NM_031966.3 (nucleotide sequenceencoding the Homo sapiens Cyclin B1); (5) GenBank Accession No. NP758505.2 (Mus musculus 430 amino acid cylcin B1); (6) GenBank AccessionNo. NM_172301.3 (nucleotide sequence encoding Mus musculus Cyclin B1).

In some embodiments, a suitable CCNB1 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 3 or SEQ ID NO. 9.

In some embodiments, a suitable CCNB1 nucleic acid sequence comprises anucleotide sequence encoding a CCNB1 polypeptide, where in someembodiments, a suitable CCNB1 polypeptide comprises an amino acidsequence encoding a polypeptide comprises a sequence having at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, or 100% amino acid sequence identity of SEQ ID NO. 4or SEQ ID NO. 10.

In some embodiments, a suitable CCND1 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 15 or SEQ ID NO. 19.

In some embodiments, a suitable CCNB1 nucleic acid sequence comprises anucleotide sequence encoding a CCNB1 polypeptide. In some embodiments, asuitable CCNB1 polypeptide comprises an amino acid sequence encoding apolypeptide comprises a sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,or 100% amino acid sequence identity of SEQ ID NO. 16 or SEQ ID NO. 20.

Aurora Kinases

Aurora kinases are serine/threonine kinases that are crucial for cellcycle control. Fu et al. (2007) Mol Cancer Res 5(1):1-10. These kinasesfunction to control chromatid segregation during cell division. To date,three mammalian aurora kinases have been identified—aurora kinase A(AURKA), aurora kinase B (AURKB) and aurora kinase C (AURKC). Inparticular, AURKB specifically functions, in part, in the attachment ofmitotic spindles to the centromere.

Amino acid sequences for aurora kinase polypeptides (e.g., AURKB) andnucleotide sequences encoding aurora kinase polypeptides (e.g., AURKB),from a variety of species, are known in the art. See, e.g.: (1) GenBankAccession No. NP 004208.2 (Homo sapiens 344 amino acid aurora kinase Bisoform 1); (2) GenBank Accession No. NP_001243763.1 (Homo sapiens 303amino acid aurora kinase B isoform 2); (3) GenBank Accession No.NP_001271455.1 (Homo sapiens 345 amino acid aurora kinase B isoform 3);(4) GenBank Accession No. AAH00442.3 (Homo sapiens 344 amino acid aurorakinase B); (5) GenBank Accession No. NM_004217.3 (nucleotide sequenceencoding the Homo sapiens AURKB, transcript variant 1); (6) GenBankAccession No. NM_001256834.1 (nucleotide sequence encoding the Homosapiens AURKB, transcript variant 2); (7) GenBank Accession No.NM_001284526.1 (nucleotide sequence encoding the Homo sapiens AURKB,transcript variant 3); (8) GenBank Accession No. NP_035626.1 (Musmusculus 345 amino acid aurora kinase B); (9) GenBank Accession No.AAH03261.1 (Mus musculus 345 amino acid aurora kinase B); (10) GenBankAccession No. NM_011496.1 (nucleotide sequence encoding the Mus musculusaurora kinase B).

In some embodiments, a suitable AURKB nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 5 or SEQ ID NO. 11.

In some embodiments, a suitable AURKB nucleic acid sequence comprises anucleotide sequence encoding an AURKB polypeptide, where in someembodiments, a suitable AURKB polypeptide comprises an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, or 100% amino acid sequenceidentity of SEQ ID NO. 6 or SEQ ID NO. 12.

It has further been discovered that introduction of nucleic acidsequences encoding CDK1, CCNB1, CDK4 and CCND1 is sufficient to inducepostmitotic cell (e.g., cardiomyocyte and neuron) proliferation and/orcell cycle reentry. In some embodiments, the CDK is CDK1 and the cyclinis CCNB1.

It has been discovered that introduction of nucleic acid sequencesencoding CDK1, CCNB1 and AURKB is sufficient to induce cardiac cell(e.g., postmitotic cardiomyocytes) proliferation and/or cell cyclereentry.

Numerous markers and methods can be used to identify postmitotic cellsthat have been induced to proliferate and/or reenter the cell cycle. Forexample, proliferating cells can be identified by immunocytochemistryanalysis for cells expressing both a proliferation marker (e.g., Ki67,PHH3, EdU) and a cell specific marker. Cells with no or lowproliferative capacity can be identified as having low or no expressionof proliferation markers. Low expression can be less than 75%, less than70%, less than 65%, less than 60%, less than 55%, less than 50%, lessthan 45%, less than 40%, less than 35%, less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, less than 5%, or noexpression as compared to a proliferating control sample (e.g., a stemor progenitor cell). Non-limiting examples of cardiac markers includecardiac troponin T (cTnT), myosin heavy chain (MYH), alpha actininand/or connexin 43. Non-limiting examples of neural marker includenestin, neuronal nuclei (NeuN), microtubule-associate protein 2 (MAP2),beta III tubulin, neuron specific enolase (NSE), oligodendrocyte lineage(Olig1/2), and glial fibrillary acidic protein (GFAP). Non-limitingexamples of pancreatic markers include Pax4, Nkx2.2, Ngn3, insulin,glucagon, and somatostatin.

The expression of various cell markers may be detected by conventionalbiochemical or immunochemical methods (e.g., enzyme-linked immunosorbentassay, immunohistochemical assay, and the like). Alternatively,expression of a nucleic acid encoding a cell specific marker can beassessed. Expression of cell-specific marker-encoding nucleic acids in acell can be confirmed by reverse transcriptase polymerase chain reaction(RT-PCR) or hybridization analysis, molecular biological methods whichhave been commonly used in the past for amplifying, detecting andanalyzing mRNA coding for any marker proteins. Nucleic acid sequencescoding for markers specific to cardiomyocytes, neural cells, pancreaticcells, hair cells, and skeletal muscle cells are known and are availablethrough public databases such as GenBank. Thus, marker-specificsequences needed for use as primers or probes are easily determined.

Genetic Modification

In some embodiments postmitotic cells (e.g., postmitotic cardiomyocytes)are induced to proliferate and/or reenter the cell cycle byadministering to the postmitotic cells a composition that geneticallymodifies the postmitotic cells. The composition may comprise at leastone nucleic acid comprising nucleotide sequences encoding a cell cycleregulating polypeptide, for example, a cyclin-dependent kinase, acyclin, and an aurora kinase polypeptide (referred to generically as “atleast one nucleic acid”). In some embodiments, an aurora kinase isexpressly excluded from the composition and/or methods of inducingproliferation and/or cell cycle reentry of the postmitotic cell.

The at least one nucleic acid comprising nucleotide sequences encodingthe proliferation and/or cell cycle reentry factors can be a recombinantexpression vector, where suitable vectors include, e.g., recombinantretroviruses, lentiviruses, and adenoviruses, adeno-associated viruses(AAV), retroviral expression vectors, lentiviral expression vectors,nucleic acid expression vectors, and plasmid expression vectors. In somecases, the at least one nucleic acid is integrated into the genome of apostmitotic cell (e.g., postmitotic cardiomyocyte) and its progeny. Inother cases, the at least one nucleic acid persists in an episomal statein the postmitotic cell and its progeny. In some cases, an endogenous,natural version of at least one proliferation and/or cell cycle reentryfactor may already exist in the postmitotic cell but an additionalexogenous factor (e.g., at least one nucleic acid) is added to thepostmitotic cell to induce proliferation and/or cell cycle reentry ofthe postmitotic cell. In other cases, the at least one nucleic acidencodes at least one proliferation and/or cell cycle reentry factorpolypeptide having an amino acid sequence that differs by one or moreamino acids from a polypeptide encoded by an endogenous proliferationand/or cell cycle reentry factor encoding nucleic acid within thepostmitotic cell.

In some embodiments, a postmitotic cell (e.g., postmitoticcardiomyocyte) is genetically modified with separate expressionconstructs, each expression construct comprising a nucleic sequenceencoding one proliferation and/or cell cycle reentry factor. In someembodiments, a postmitotic cell (e.g., postmitotic cardiomyocyte) isgenetically modified with multiple expression constructs, eachexpression construct comprising a nucleic sequence encoding oneproliferation and/or cell cycle reentry factor. In some embodiments, apostmitotic cell (e.g., postmitotic cardiomyocyte) is geneticallymodified with three separate expression constructs, each expressionconstruct comprising a nucleic sequence encoding one proliferationand/or cell cycle reentry factor. In some embodiments, a postmitoticcell (e.g., postmitotic cardiomyocyte) is genetically modified with twoseparate expression constructs, each expression construct comprising anucleic sequence encoding at least one proliferation and/or cell cyclereentry factor. In some embodiments, a postmitotic cell (e.g.,postmitotic cardiomyocyte) is genetically modified with one expressionconstruct, the expression construct comprising a nucleic sequenceencoding at least one proliferation and/or cell cycle reentry factor. Insome embodiments, a postmitotic cell (e.g., postmitotic cardiomyocyte)is genetically modified with one expression construct, the expressionconstruct comprising nucleotide sequences encoding sequences of severalcell cycle regulating factors. In some embodiments, a postmitotic cell(e.g., postmitotic cardiomyocyte) is genetically modified with oneexpression construct, the expression construct comprising nucleotidesequences encoding sequences of all of CDK1 and CCNB1, and optionallyincluding CDK4 and/or CCND1. In some embodiments, sequence encoding CDK1and CCNB1, and optionally including CDK4 and/or CCND1, are in oneexpression vector, two expression vectors (e.g., CDK1 is expressed froma separate vector as CCNB1), or four expression vectors (e.g., CDK1,CCNB1, CDK4 and CCND1 are all expressed from separate vectors).

In some embodiments, a postmitotic cell (e.g., postmitoticcardiomyocyte) is genetically modified with one expression construct,the expression construct comprising nucleotide sequences encodingsequences of all three of CDK1, CCNB1 and AURKB. In some embodiments,sequence encoding CDK1, CCNB1 and AURKB are in one expression vector,two expression vectors (e.g., CDK1 is expressed from a separate vectoras CCNB1 and AURKB), or three expression vectors (e.g., CDK1, CCNB1 andAURKB are all expressed from separate vectors).

In some embodiments, one or more exogenous nucleic acids comprisingnucleotide sequences encoding proliferation and/or cell cycle reentryfactor polypeptides is introduced into a single postmitotic cell (e.g.,postmitotic cardiomyocyte). In other embodiments, one or more exogenousnucleic acids comprising nucleotide sequences encoding proliferationand/or cell cycle reentry factor polypeptides is introduced into apopulation of postmitotic cells (e.g., a population of postmitoticcardiomyocytes) in vitro. In some embodiments, one or more exogenousnucleic acids comprising nucleotide sequences encoding proliferationand/or cell cycle reentry factor polypeptides is introduced into apostmitotic cell (e.g., a single postmitotic cardiomyocyte or apopulation of postmitotic cardiomyocytes) in vivo.

Where a population of cardiac cells is genetically modified (in vitro orin vivo) with one or more exogenous nucleic acids comprising nucleotidesequences encoding proliferation and/or cell cycle reentry factorpolypeptides, the one or more exogenous nucleic acids can be introducedinto greater than 0.1% of the total population of postmitotic cells,e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or otherpercent of cells greater than 0.1%.

In other embodiments, where a population of postmitotic cells isgenetically modified (in vitro or in vivo) with one or more exogenousnucleic acids comprising nucleotide sequences encoding proliferationand/or cell cycle reentry factor polypeptides, the one or more exogenousnucleic acids can be introduced into greater than 1% of the totalpopulation of cardiac cells, e.g., 2%, 3%, 5%, 10%, 12%, 15%, 20%, orother percent of cells greater than 1%.

In other embodiments, where a population of postmitotic cells isgenetically modified (in vitro or in vivo) with one or more exogenousnucleic acids comprising nucleotide sequences encoding proliferationand/or cell cycle reentry factor polypeptides, the one or more exogenousnucleic acids can be introduced into greater than 20% of the totalpopulation of postmitotic cells, e.g., 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or other percent of cellsgreater than 20%.

In some embodiments, the one or more nucleic acids comprising nucleotidesequences encoding proliferation and/or cell cycle reentry factorpolypeptides is in an expression construct that provides for productionof the one or more proliferation and/or cell cycle reentry factorpolypeptides in the genetically modified postmitotic cells. In someembodiments, the expression construct is a viral construct, e.g., arecombinant adeno-associated virus construct (e.g., U.S. Pat. No.7,078,387), a recombinant adenoviral construct, a recombinant lentiviralconstruct, etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (e.g., Li et al. (1994) Invest Opthalmol Vis Sci35:2543-2549; Borras et al. (1999) Gene Ther 6:515-524; Li and Davidson,(1995) Proc. Natl. Acad. Sci. 92:7700-7704; Sakamoto et al. (1999) HumGene Ther 5:1088-1097; WO 94/12649; WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (e.g.,Ali et al. (1998) Hum Gene Ther 9(1):81-86, 1998, Flannery et al. (1997)Proc. Natl. Acad. Sci. 94:6916-6921; Bennett et al. (1997) InvestOpthalmol Vis Sci 38:2857-2863; Jomary et al. (1997) Gene Ther4:683-690; Rolling et al. (1999), Hum Gene Ther 10:641-648; Ali et al.(1996) Hum Mol Genet. 5:591-594; WO 93/09239, Samulski et al. (1989) J.Vir. 63:3822-3828; Mendelson et al. (1988) Virol. 166:154-165; andFlotte et al. (1993) Proc. Natl. Acad. Sci. 90:10613-10617; SV40; herpessimplex virus; human immunodeficiency virus (e.g., Miyoshi et al. (1997)Proc. Natl. Acad. Sci. 94:10319-10323; Takahashi et al. (1999) J Virol73:7812-7816); a retroviral vector (e.g., Murine-Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), and pAd (LifeTechnologies). However, any other vector may be used so long as it iscompatible with the cells of the present disclosure.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a proliferation and/or cell cycle reentry factorencoding nucleotide sequence (e.g., a CCNB1-encoding nucleotidesequence, an AURKB-encoding nucleotide sequence, a CDK1-encodingnucleotide sequence, a CCND1-encoding nucleotide sequence, or aCDK4-encoding nucleotide sequence) is operably linked to a controlelement, e.g., a transcriptional control element, such as a promoter.The transcriptional control element is functional in a eukaryotic cell,for example, a mammalian cardiac cell. Suitable transcriptional controlelements include promoters and enhancers. In some embodiments, thepromoter is constitutively active. In other embodiments, the promoter isinducible.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include CMV, CMV immediate early, HSVthymidine kinase, early and late SV40, long terminal repeats (LTRs) fromretrovirus, and mouse metallothionein-I. In some embodiments, promotersthat are capable of conferring cardiac specific expression will be used.Non-limiting examples of suitable cardiac specific promoters includedesmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2(MLC-2), cardiac troponin T (cTnT) and cardiac troponin C (cTnC).Non-limiting examples of suitable neuron specific promoters includesynapsin I (SYN), calcium/calmodulin-dependent protein kinase II,tubulin alpha I, neuron-specific enolase and platelet-derived growthfactor beta chain promoters and hybrid promoters by fusingcytomegalovirus enhancer (E) to those neuron-specific promoters.

In some embodiments, a proliferation and/or cell cycle reentry factorencoding nucleotide sequence is operably linked to a cell type-specifictranscriptional regulator element (TRE), where TREs include promotersand enhancers. Suitable TREs include, but are not limited to, TREsderived from the following genes: myosin light chain-2, α-myosin heavychain, AE3, cardiac troponin C, and cardiac actin. Franz et al. (1997)Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci.752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al.(1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA89:4047-4051.

Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. The expression vector may alsocontain a ribosome binding site for translation initiation and atranscription terminator. The expression vector may also includeappropriate sequences for amplifying expression.

Examples of suitable mammalian expression vectors (expression vectorssuitable for use in mammalian postmitotic cells) include, but are notlimited to: recombinant viruses, nucleic acid vectors, such as plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, humanartificial chromosomes, cDNA, cRNA, and polymerase chain reaction (PCR)product expression cassettes. Examples of suitable promoters for drivingexpression of proliferation and/or cell cycle reentry factorpolypeptide-encoding nucleotide sequences include, but are not limitedto, retroviral long terminal repeat (LTR) elements; constitutivepromoters such as CMV, HSV1-TK, SV40, EF-1α, β-actin, phosphoglycerolkinase (PGK); inducible promoters, such as those containing Tet-operatorelements; cardiac specific promoters, such as desmin (Des), alpha-myosinheavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin T(cTnT) and cardiac troponin C (cTnC); neural specific promoters, such asnestin, neuronal nuclei (NeuN), microtubule-associate protein 2 (MAP2),beta III tubulin, neuron specific enolase (NSE), oligodendrocyte lineage(Olig1/2), and glial fibrillary acidic protein (GFAP); and pancreaticspecific promoters, such as Pax4, Nkx2.2, Ngn3, insulin, glucagon, andsomatostatin.

In some cases, the mammalian expression vector(s) encodes, in additionto exogenous proliferation and/or cell cycle reentry factorpolypeptides, a marker gene that facilitates identification or selectionof cells that have been transfected, transduced or infected. Examples ofmarker genes include, but are not limited to, genes encoding fluorescentproteins, e.g., enhanced green fluorescent protein, Ds-Red (DsRed:Discosoma sp. red fluorescent protein (RFP); Bevis et al. (2002) Nat.Biotechnol. 20(11):83-87), yellow fluorescent protein, mCherry, andcyanofluorescent protein; and genes encoding proteins conferringresistance to a selection agent, e.g., a neomycin resistance gene, apuromycin resistance gene, a blasticidin resistance gene, and the like.

In one embodiment, the expression vector further comprises, oralternatively consists essentially of, or yet further consists of asuicide gene. Expression of the suicide gene may be regulated by thesame or different promoter as that which expresses the at least oneproliferation and/or cell cycle reentry factor polypeptide-encodingnucleotide. A suicide gene is one that allows for the negative selectionof the cells. In the methods described herein, a suicide gene is used asa safety system, allowing the cells expressing the gene to be killed byintroduction of a selective agent. This is desirable in case therecombinant gene causes a mutation leading to uncontrolled cell growth.A number of suicide gene systems have been identified, including theherpes simplex virus thymidine kinase (tk or TK) gene, the cytosinedeaminase gene, the varicella-zoster virus thymidine kinase gene, thenitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deogene (also see, for example, Yazawa K, Fisher W E, Brunicardi F C:Current progress in suicide gene therapy for cancer. World J. Surg.(2002) 26(7):783-9). In one embodiment, the suicide gene is thethymidine kinase (TK) gene. In one aspect, the TK gene is a wild-type TKgene. In other aspects, the TK gene is a mutated form of the gene, e.g.,sr23tk. Cells expressing the TK protein can be killed using ganciclovir.In another embodiment, the nucleic acid encoding the tetracyclineactivator protein and the suicide gene are regulated by one promoter.

Examples of suitable viral vectors include, but are not limited, viralvectors based on retroviruses (including lentiviruses); adenoviruses;adeno-associated viruses, and episomal vectors. An example of a suitableretrovirus-based vector is a vector based on murine moloney leukemiavirus (MMLV); however, other recombinant retroviruses may also be used,e.g., Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus(MLV), Mink-Cell focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, Mason PfizerMonkey Virus, or Rous Sarcoma Virus (e.g., U.S. Pat. No. 6,333,195).

In other cases, the retrovirus-based vector is a lentivirus-basedvector, (e.g., Human Immunodeficiency Virus-1 (HIV-1), SimianImmunodeficiency Virus (SIV) or Feline Immunodeficiency Virus (FIV)).Johnston et al. (1999), Journal of Virology, 73(6):4991-5000 (FIV);Negre D et al. (2002) Current Topics in Microbiology and Immunology,261:53-74 (SIV); Naldini et al. (1996) Science, 272:263-267 (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g.,retroviral env) to aid entry into the target cell. Such viralpolypeptides are well-established in the art, for example, U.S. Pat. No.5,449,614. The viral polypeptide may be an amphotropic viralpolypeptide, for example, amphotropic env, which aids entry into cellsderived from multiple species, including cells outside of the originalhost species. The viral polypeptide may be a xenotropic viralpolypeptide that aids entry into cells outside of the original hostspecies. In some embodiments, the viral polypeptide is an ecotropicviral polypeptide, for example, ecotropic env, which aids entry intocells of the original host species.

Examples of viral polypeptides capable of aiding entry of retrovirusesinto cells include but are not limited to: MMLV amphotropic env, MMLVecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein(VSV-g), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C,FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. Yeeet al. (1994) Methods Cell Biol., Pt A:99-112 (VSV-G); U.S. Pat. No.5,449,614. In some cases, the viral polypeptide is genetically modifiedto promote expression or enhanced binding to a receptor.

In general, a recombinant virus is produced by introducing a viral DNAor RNA construct into a producer cell. In some cases, the producer celldoes not express exogenous genes. In other cases, the producer cell is a“packaging cell” comprising one or more exogenous genes, e.g., genesencoding one or more gag, pol, or env polypeptides and/or one or moreretroviral gag, pol, or env polypeptides. The retroviral packaging cellmay comprise a gene encoding a viral polypeptide, e.g., VSV-g that aidsentry into target cells. In some cases, the packaging cell comprisesgenes encoding one or more lentiviral proteins, e.g., gag, pol, env,vpr, vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cellcomprises genes encoding adenovirus proteins such as E1A or E1B or otheradenoviral proteins. For example, proteins supplied by packaging cellsmay be retrovirus-derived proteins such as gag, pol, and env;lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif,tat, rev, and nef; and adenovirus-derived proteins such as E1A and E1B.In many examples, the packaging cells supply proteins derived from avirus that differs from the virus from which the viral vector derives.

Packaging cell lines include but are not limited to anyeasily-transfectable cell line. Packaging cell lines can be based on293T cells, NIH3T3, COS or HeLa cell lines. Packaging cells are oftenused to package virus vector plasmids deficient in at least one geneencoding a protein required for virus packaging. Any cells that cansupply a protein or polypeptide lacking from the proteins encoded bysuch virus vector plasmid may be used as packaging cells. Examples ofpackaging cell lines include but are not limited to: Platinum-E(Plat-E), Platinum-A (Plat-A), BOSC 23 (ATCC CRL 11554) and Bing (ATCCCRL 11270). Morita et al. (2000) Gene Therapy 7(12):1063-1066; Onishi etal. (1996) Experimental Hematology, 24:324-329; U.S. Pat. No. 6,995,009.Commercial packaging lines are also useful, e.g., Ampho-Pak 293 cellline, Eco-Pak 2-293 cell line, RetroPack PT67 cell line, and Retro-XUniversal Packaging System (all available from Clontech).

The retroviral construct may be derived from a range of retroviruses,e.g., MMLV, HIV-1, SIV, FIV, or other retrovirus described herein. Theretroviral construct may encode all viral polypeptides necessary formore than one cycle of replication of a specific virus. In some cases,the efficiency of viral entry is improved by the addition of otherfactors or other viral polypeptides. In other cases, the viralpolypeptides encoded by the retroviral construct do not support morethan one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In suchcircumstances, the addition of other factors or other viral polypeptidescan help facilitate viral entry. In an exemplary embodiment, therecombinant retrovirus is HIV-1 virus comprising a VSV-g polypeptide butnot comprising a HIV-1 env polypeptide.

The retroviral construct may comprise: a promoter, a multi-cloning site,and/or a resistance gene. Examples of promoters include but are notlimited to CMV, SV40, EF1α, β-actin; retroviral LTR promoters, andinducible promoters. The retroviral construct may also comprise apackaging signal (e.g., a packaging signal derived from the MFG vector;a psi packaging signal). Examples of some retroviral constructs known inthe art include but are not limited to: pMX, pBabeX or derivativesthereof. Onishi et al. (1996) Experimental Hematology, 24:324-329. Insome cases, the retroviral construct is a self-inactivating lentiviralvector (SIN) vector. Miyoshi et al. (1998) J. Virol. 72(10):8150-8157.In some cases, the retroviral construct is LL-CG, LS-CG, CL-CG, CS-CG,CLG or MFG. Miyoshi et al. (1998) J Virol. 72(10):8150-8157; Onishi etal. (1996) Experimental Hematology, 24:324-329; Riviere et al. (1995)Proc. Natl. Acad. Sci., 92:6733-6737. Virus vector plasmids (orconstructs), include: pMXs, pMxs-IB, pMXs-puro, pMXs-neo (pMXs-IB is avector carrying the blasticidin-resistant gene instead of thepuromycin-resistant gene of pMXs-puro) Kimatura et al. (2003)Experimental Hematology 31: 1007-1014; MFG Riviere et al. (1995) Proc.Natl. Acad. Sci., 92:6733-6737; pBabePuro; Morgenstern et al. (1990)Nucleic Acids Research 18:3587-3596; LL-CG, CL-CG, CS-CG, CLG Miyoshi etal. (1998) J Vir. 72:8150-8157 and the like as the retrovirus system,and pAdexl Kanegae et al. (1995) Nucleic Acids Research 23:3816-3821 andthe like as the adenovirus system. In exemplary embodiments, theretroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin(e.g., pMXs-puro, pBabePuro); or neomycin (e.g., pMXs-neo). Morgensternet al. (1990) Nucleic Acids Research 18:3587-3596.

Methods of producing recombinant viruses from packaging cells and theiruses are well established; see, e.g., U.S. Pat. Nos. 5,834,256;6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009. Many methodsbegin with the introduction of a viral construct into a packaging cellline. The viral construct may be introduced into a host fibroblast byany method known in the art, including but not limited to: a calciumphosphate method, a lipofection method (e.g., Felgner et al. (1987)Proc. Natl. Acad. Sci. 84:7413-7417), an electroporation method,microinjection, Fugene transfection, nucleofection and the like, and anymethod described herein.

One or more nucleic acids encoding proliferation and/or cell cyclereentry factors can be introduced into a postmitotic cell using avariety of well-known techniques, such as non-viral based transfectionof the cell. In an exemplary aspect a construct is incorporated into avector and introduced into a cardiac cell. Introduction into the cardiaccell may be performed by any non-viral based transfection method knownin the art, such as, but not limited to, electroporation, calciumphosphate mediated transfer, nucleofection, sonoporation, heat shock,magnetofection, liposome mediated transfer, microinjection,microprojectile mediated transfer (nanoparticles), cationic polymermediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol(PEG) and the like, or cell fusion. Other methods of transfectioninclude transfection reagents such as Lipofectamine™ Dojindo Hilymax™,Fugene™, jetPEI™, Effectene™, and DreamFect™.

In some embodiments, the at least one nucleic acid is a syntheticmessenger RNA (mRNA). Synthetic mRNAs provide the genetic informationfor making proteins of interest and can be chemically modified to avoidtriggering an immune response. Zangi et al. (2013) Nature Biotech31:898-907. Since mRNAs do not integrate into the host cell genome, thesynthetic RNA acts for a period of time and then disappears as the celldivides. In some embodiments the synthetic mRNAs are modified, forexample, with pseudouridine and/or 5-methyl-cytidine, to reduce innateantiviral response to single-stranded RNA. In some embodiments, thesynthetic RNAs encode CCNB1, CCND1, CDK1, CDK4, AURKB or combinationsand/or equivalents of each thereof.

In some embodiments, a suitable synthetic RNA encoding CDK1 comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 1 or SEQ ID NO. 7. In someembodiments, a suitable synthetic RNA encoding CCNB1 comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 3 or SEQ ID NO. 9. In someembodiments, a suitable synthetic RNA encoding AURKB comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 5 or SEQ ID NO. 11. In someembodiments, a suitable synthetic RNA encoding CDK4 comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 13 or SEQ ID NO. 17. In someembodiments, a suitable synthetic RNA encoding CCND1 comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%nucleotide sequence identity of SEQ ID NO. 15 or SEQ ID NO. 19.

Protein Modification

In another aspect polypeptides can be introduced into postmitotic cellsusing any method known in the art. Protein delivery (i.e., proteintransduction) is the process by which a peptide or protein motif crossesthe cell plasma membrane. Methods to introduce peptides into cellsinclude, for example, transfection, micro-injection, electroporation,nanoparticle, virus-like particles (VLP). In some embodiments, theproteins are CCNB1, CCND1, CDK1, CDK4, AURKB or combinations and/orequivalents of each thereof.

Chemical Modification

In another aspect, the disclosure provides a method of inducingproliferation and/or cell cycle reentry of a postmitotic cells, themethod comprising administering to the cell a composition comprising atleast one chemical in an amount effective to stimulate proliferationand/or cell cycle reentry wherein said chemical composition is capableof increasing expression of a cyclin-dependent kinase, a cyclin, and anaurora kinase, or equivalents thereof. In other embodiments, the methodcomprises, or alternatively consists essentially of, or yet furtherconsists of, contacting the cell a with an effective amount of at leastone chemical that increases expression of at least one cyclin-dependentkinase, at least one a cyclin, or equivalents thereof. In someembodiments, the chemical increases expression of CCNB1, CCND1, CDK1,CDK4, combinations and/or equivalents of each thereof.

Any compound now known or later discovered which is capable ofincreasing expression of a cyclin-dependent kinase, a cyclin, or anaurora kinase (e.g., CDK1, CDK4, CCNB1, CCND1, or AURKB) or equivalentsof each thereof is within the purview of this disclosure. Compoundsshould be understood to also encompass all pharmaceutically acceptablederivatives that can be used in association with one or morepharmaceutically acceptable excipients, diluents or carriers.

In other aspects, the disclosure provides a method of inducingproliferation and/or cell cycle reentry of a postmitotic cell, themethod comprising, or alternatively consisting essentially of, or yetfurther consisting of, contacting the cell with an effective amount of achemical composition to stimulate proliferation and/or cell cyclereentry wherein said chemical composition is capable of inhibitingexpression of negative regulators of cellular proliferation and/or cellcycle reentry. For example, a Weel inhibitor can be used to induceexpression of CDK1. In some embodiments, activation of CDK1 with smallmolecule MK1775 (a Weel inhibitor) can replace CDK1 and/or CCNB from thecocktail.

Additional Agents and Cell Cycle Regulating Factors

A postmitotic cell (e.g., a postmitotic cardiomyocyte) can be modified(genetically or non-genetically) as described above, and can also becontacted with one or more additional agents and/or factors which can beadded to the composition, e.g., the one or more additional agents and/orfactors can be included as additives to the culture media or to thecomposition.

In some embodiments, other cell cycle regulating genes can be includedin the composition including, for example, actin binding protein anillin(ANLN), cell division cycle-5 (CDC5), cadherin-6 (CDH6), cyclindependent-kinase 2 (CDK2), cyclin dependent-kinase 3 (CDK3), centromereprotein A (CENPA), COPS signalosome subunit 5 (COPS5), cullin 3 (CUL3),GTPase-activating protein (CYK4), protein regulator of cytokinesis 1(PRC1) and/or WNT1-inducible signaling pathway protein 1 (WISP1).

In some embodiments, use of a compound that releases cell cycle blocksin postmitotic cells, for example a p38 inhibitor, a p21 inhibitor, ap57 inhibitor, or a pharmaceutically acceptable derivative thereof canbe used to facilitate an increase in proliferation and/or cell cyclereentry. In some embodiments, a small molecule inhibitor can be used. Inother embodiments, an siRNA can be used to decrease expression. Bothsmall molecule inhibitors and siRNAs for p38, p21, p57 and the like arecommercially available.

In some embodiments, a first composition of one or more proliferationand/or cell cycle reentry factors is added to a postmitotic cell for aperiod of time followed by a second composition of one or moreproliferation and/or cell cycle reentry factors. For example, acombination of CDK4 and CCND1 can act to enhance the number of cells inG1 and S, referred to herein as a “G1 cocktail.” Other combinations offactors, for example, CDK1 and CCNB can act as a “G2 cocktail” andenhance the number of cells in G2, but not G1 or S. Combinations ofCDK1, CCNB1, CDK4 and CCND1 can induce a balanced distribution of G1/G2cells. In some embodiments, a G1 cocktail is added for a period of time,followed by a G2 cocktail. The G2 cocktail can be added to the G1cocktail or can replace the G2 cocktail. In some embodiments, after theG2 cocktail has been added to the postmitotic cells for a period oftime, the G1 cocktail can be added to the cells. The G1 cocktail can beadded in addition to the G2 cocktail, or the G1 cocktail can replace theG2 cocktail. In one preferred embodiment, a G1 cocktail of CDK4 andCCND1 modified mRNAs can be added to a population of postmitotic cellsfor a period of time, the G1 cocktail can then be removed, and a G2cocktail of CDK1 and CCNB1 modified mRNAs can be added to the populationof postmitotic cells for a period of time. The G1 and G2 cocktails canbe alternated in a similar pattern to induce proliferation of thepostmitotic cells. It is contemplated that alternating exposure of thepostmitotic cells to the G1 and G2 cocktails can help to moreefficiently drive the cells through the cell cycle.

III. Compositions

The present disclosure also provides an isolated, proliferativepostmitotic cell modified to overexpress proliferation and/or cell cyclereentry factors (e.g., CDK1, CCNB1, and AURKB or equivalents thereof).

In one aspect the disclosure also provides a composition comprising, oralternatively consisting essentially or, or yet further consisting of,an isolated, proliferative postmitotic cell modified to overexpressproliferation and/or cell cycle reentry factors (e.g., CDK1, CCNB1,CDK4, CCND1, and/or AURKB or combinations and/or equivalents of eachthereof).

In some aspects are provided, isolated, proliferative postmitotic cellmodified to overexpress at least one CDK and at least one cyclin, or anequivalent of each thereof. In other aspects are provided isolated,proliferative postmitotic cell modified to overexpress CDK1 and CCNB1,or an equivalent of each thereof. In some embodiments, the cells arefurther modified to overexpress CDK4, CCND1, or both.

In another aspect provided is a substantially homogenous populationproliferative postmitotic cells. In some embodiments, a compositioncomprises a population of proliferative postmitotic cells describedherein and a carrier, optionally a pharmaceutically acceptableexcipient. In some embodiments, the compositions further comprise astabilizer and/or a preservative.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The composition may comprise a pharmaceutically acceptable excipient, apharmaceutically acceptable salt, diluents, carriers, vehicles and suchother inactive agents well known to the skilled artisan. Vehicles andexcipients commonly employed in pharmaceutical preparations include, forexample, talc, gum Arabic, lactose, starch, magnesium stearate, cocoabutter, aqueous or non-aqueous solvents, oils, paraffin derivatives,glycols, etc. Solutions can be prepared using water or physiologicallycompatible organic solvents such as ethanol, 1,2-propylene glycol,polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partialesters of glycerine and the like. Parenteral compositions may beprepared using conventional techniques that may include sterile isotonicsaline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol,polyglycols mixed with water, Ringer's solution, etc. In one aspect, acoloring agent is added to facilitate in locating and properly placingthe composition to the intended treatment site.

Compositions may include a preservative and/or a stabilizer.Non-limiting examples of preservatives include methyl-, ethyl-,propyl-parabens, sodium benzoate, benzoic acid, sorbic acid, potassiumsorbate, propionic acid, benzalkonium chloride, benzyl alcohol,thimerosal, phenylmercurate salts, chlorhexidine, phenol, 3-cresol,quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, andimidurea.

To control tonicity, the composition can comprise a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride and calcium chloride.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer; or a citrate buffer. Buffers will typically beincluded at a concentration in the 5-20 mM range. The pH of acomposition will generally be between 5 and 8, and more typicallybetween 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8.

The composition can be administered by any appropriate route, which willbe apparent to the skilled person depending on the disease or conditionto be treated. Typical routes of administration include intravenous,intra-arterial, intramuscular, subcutaneous, intracranial, intranasal orintraperitoneal.

In some embodiments, the composition may include a cryoprotectant agent.Non-limiting examples of cryoprotectant agents include a glycol (e.g.,ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide(DMSO), formamide, sucrose, trehalose, dextrose, and any combinationsthereof.

The composition can be included in an implantable device. Suitableimplantable devices contemplated by this invention include intravascularstents (e.g., self-expandable stents, balloon-expandable stents, andstent-grafts), scaffolds, grafts, and the like. Such implantable devicescan be coated on at least one surface, or impregnated, with acomposition capable of inducing cell cycle reentry of a postmitoticcell. The composition may also be contained within a reservoir in theimplantable device. Where the composition is contained within areservoir in the implantable device, the reservoir is structured so asto allow the composition to elute from the device. The composition maycomprise CDK1, CDK4, CCNB1, CCND1, and AURKB polypeptides, or anycombination or equivalents of each thereof. The present disclosurefurther provides an implantable device that comprises a compositioncomprising nucleotide sequences encoding CDK1, CDK4, CCNB1, CCND1, andAURKB polypeptides, or any combination or equivalents of each thereof.

The composition can comprise a pharmaceutically acceptable excipient, apharmaceutically acceptable salt, diluents, carriers, vehicles and suchother inactive agents well known to the skilled artisan. Vehicles andexcipients commonly employed in pharmaceutical preparations include, forexample, talc, gum Arabic, lactose, starch, magnesium stearate, cocoabutter, aqueous or non-aqueous solvents, oils, paraffin derivatives,glycols, etc. Solutions can be prepared using water or physiologicallycompatible organic solvents such as ethanol, 1,2-propylene glycol,polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partialesters of glycerine and the like. Parenteral compositions may beprepared using conventional techniques that may include sterile isotonicsaline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol,polyglycols mixed with water, Ringer's solution, etc. In one aspect, acoloring agent is added to facilitate in locating and properly placingthe composition to the intended treatment site.

In some embodiments, the formulation is a controlled releaseformulation. The term “controlled release formulation” includessustained release and time-release formulations. Controlled releaseformulations are well-known in the art. These include excipients thatallow for sustained, periodic, pulse, or delayed release of thecomposition. Controlled release formulations include, withoutlimitation, embedding of the composition (cells and/or at least oneproliferation and/or cell cycle reentry factor) into a matrix; entericcoatings; micro-encapsulation; gels and hydrogels; implants; and anyother formulation that allows for controlled release of a composition.

In one aspect is provided a kit of parts comprising the above-mentionedcomposition (cells and/or at least one proliferation and/or cell cyclereentry factor), reagents and culture medium. The kit may furthercomprise a document or an instruction that describes a protocol forgrowing the cells in culture or for administering to a subject in needthereof.

IV. Methods of Treatment

Cardiovascular Disease

In one aspect is provided methods for treating a cardiovascular diseasecomprising administering to a subject in need thereof, an effectiveamount of a composition that increases the expression of CDK1 and CCNB1,or an equivalent of each thereof. In some embodiments, the methodsfurther comprise administering an effective amount of a composition thatincreases the expression of CDK4, CCND1, or both.

In another aspect is provided methods for treating a cardiovasculardisease comprising administering to a subject in need thereof, aneffective amount of a population of the proliferative postmitoticcardiac cells (e.g., cardiomyocytes) disclosed herein. In someembodiments, the proliferative cardiomyocytes are modified tooverexpress CDK1 and CCNB1, or equivalents thereof. In otherembodiments, the proliferative cardiomyocytes are modified tooverexpress CDK4, CCND1, or both.

In another aspect is provided methods of treating a cardiovasculardisease by administering to a subject in need thereof, an effectiveamount of a composition comprising proliferation and/or cell cyclereentry factors (e.g., CDK1, CCNB1 and AURKB or equivalents thereof).

In some aspects, a proliferative cardiac cell (e.g., a cardiomyocyte) ofthe present disclosure can be used to treat a subject in need thereof.In some embodiments, the proliferative cardiac cell can be administeredto the subject in need thereof, where administration into the subject ofthe proliferative cardiac cell, treats a cardiovascular disease in thesubject. Thus, in some embodiments, a method of treating cardiovasculardisease involves administering to a subject in need thereof a populationof cardiac cells induced to proliferate and/or reenter the cell cycle.In other embodiments, a method of treating cardiovascular diseaseinvolves administering to the subject in need thereof an effectiveamount of a composition comprising CDK1, CCNB1 and AURKB or equivalentsthereof. In other embodiments, a method of treating cardiovasculardisease involves administering to the subject in need thereof aneffective amount of a composition comprising CDK1, CDK4, CCNB1, CCND1,or combinations and/or equivalents thereof.

Subjects in need of treatment using the compositions, cells and methodsof the present disclosure include, but are not limited to, individualshaving a congenital heart defect, individuals suffering from adegenerative muscle disease, individuals suffering from a condition thatresults in ischemic heart tissue (e.g., individuals with coronary arterydisease), and the like. In some examples, a method is useful to treat adegenerative muscle disease or condition (e.g., familial cardiomyopathy,dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, or coronary artery disease with resultant ischemiccardiomyopathy). In some examples, a subject method is useful to treatindividuals having a cardiac or cardiovascular disease or disorder, forexample, cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism.

Subjects who are suitable for treatment using the compositions, cellsand methods of the present disclosure include individuals (e.g.,mammalian subjects, such as humans, non-human primates, experimentalnon-human mammalian subjects such as mice, rats, etc.) having a cardiaccondition including but limited to a condition that results in ischemicheart tissue (e.g., individuals with coronary artery disease) and thelike. In some examples, an individual suitable for treatment suffersfrom a cardiac or cardiovascular disease or condition, e.g.,cardiovascular disease, aneurysm, angina, arrhythmia, atherosclerosis,cerebrovascular accident (stroke), cerebrovascular disease, congenitalheart disease, congestive heart failure, myocarditis, valve diseasecoronary, artery disease dilated, diastolic dysfunction, endocarditis,high blood pressure (hypertension), cardiomyopathy, hypertrophiccardiomyopathy, restrictive cardiomyopathy, coronary artery disease withresultant ischemic cardiomyopathy, mitral valve prolapse, myocardialinfarction (heart attack), or venous thromboembolism. In some examples,individuals suitable for treatment with a subject method includeindividuals who have a degenerative muscle disease, e.g., familialcardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, or coronary artery disease with resultantischemic cardiomyopathy.

Neurological Disease

In one aspect is provided methods for treating a neurological diseasecomprising administering to a subject in need thereof an effectiveamount of a composition that increases the expression of CDK1 and CCNB1,or equivalents of each thereof. In some embodiments, the methods furthercomprise administering an effective amount of a composition thatincreases the expression of CDK4, CCND1, or both.

In another aspect is provided methods for treating a neurologicaldisease comprising administering to a subject in need thereof, aneffective amount of a population of the proliferative postmitotic neuralcells (e.g., neurons) disclosed herein. In some embodiments, theproliferative neural cells are modified to overexpress CDK1 and CCNB1,or equivalents thereof. In other embodiments, the proliferative neuralare modified to overexpress CDK4, CCND1, or both.

In another aspect is provided methods of treating a neurological diseaseby administering to a subject in need thereof, an effective amount of acomposition comprising proliferation and/or cell cycle reentry factors(e.g., CDK1, CCNB1 and AURKB or equivalents thereof).

In some aspects, a proliferative neural cell (e.g., a neuron) of thepresent disclosure can be used to treat a subject in need thereof. Insome embodiments, the proliferative neural cell can be administered tothe subject in need thereof, where administration into the subject ofthe proliferative neural cell, treats a neurological disease in thesubject. Thus, in some embodiments, a method of treating a neuraldisease involves administering to a subject in need thereof a populationof neural cells induced to proliferate and/or reenter the cell cycle. Inother embodiments, a method of treating a neural disease involvesadministering to the subject in need thereof an effective amount of acomposition comprising CDK1, CCNB1 and AURKB or equivalents thereof. Inother embodiments, a method of treating a neural disease involvesadministering to the subject in need thereof an effective amount of acomposition comprising CDK1, CDK4, CCNB1, CCND1, or combinations and/orequivalents thereof.

Subjects in need of or suitable for treatment using the compositions,cells and methods of the present disclosure include, but are not limitedto, individuals having a spinal cord injury (SCI), amyotrophic lateralsclerosis (ALS), dural arteriovenous fistulae, epilepsy, memorydisorders, multiple sclerosis (MS), Parkinson's disease, peripheralneuropathy, Alzheimer's disease, post-herpetic neuralgia, spinal cordtumor, and stroke.

Pancreatic Disease

In one aspect is provided methods for treating a pancreatic diseasecomprising administering to a subject in need thereof an effectiveamount of a composition that increases the expression of CDK1 and CCNB1,or equivalents of each thereof. In some embodiments, the methods furthercomprise administering an effective amount of a composition thatincreases the expression of CDK4, CCND1, or both.

In another aspect is provided methods for treating a pancreatic diseasecomprising administering to a subject in need thereof, an effectiveamount of a population of the proliferative postmitotic pancreatic cells(e.g., beta cell) disclosed herein. In some embodiments, theproliferative pancreatic cells are modified to overexpress CDK1 andCCNB1, or equivalents thereof. In other embodiments, the proliferativepancreatic cells are modified to overexpress CDK4, CCND1, or both.

In another aspect is provided methods of treating a pancreatic diseaseby administering to a subject in need thereof, an effective amount of acomposition comprising proliferation and/or cell cycle reentry factors(e.g., CDK1, CCNB1 and AURKB or equivalents thereof).

In some aspects, a proliferative pancreatic cell (e.g., a beta cell) ofthe present disclosure can be used to treat a subject in need thereof.In some embodiments, the proliferative pancreatic cell can beadministered to the subject in need thereof, where administration intothe subject of the proliferative pancreatic cell, treats a pancreaticdisease in the subject. Thus, in some embodiments, a method of treatinga pancreatic disease involves administering to a subject in need thereofa population of pancreatic cells induced to proliferate and/or reenterthe cell cycle. In other embodiments, a method of treating a pancreaticdisease involves administering to the subject in need thereof aneffective amount of a composition comprising CDK1, CCNB1 and AURKB orequivalents thereof. In other embodiments, a method of treating apancreatic disease involves administering to the subject in need thereofan effective amount of a composition comprising CDK1, CDK4, CCNB1,CCND1, or combinations and/or equivalents thereof.

Subjects in need of or suitable for treatment using the compositions,cells and methods of the present disclosure include, but are not limitedto, individuals having diabetes mellitus (e.g., type 1 diabetes or type2 diabetes), acute pancreatitis, chronic pancreatitis, hereditarypancreatitis, pancreatic cancer, cystic fibrosis, and congenitalmalformations.

Hearing Loss

In one aspect is provided methods for treating hearing loss comprisingadministering to a subject in need thereof an effective amount of acomposition that increases the expression of CDK1 and CCNB1, orequivalents of each thereof. In some embodiments, the methods furthercomprise administering an effective amount of a composition thatincreases the expression of CDK4, CCND1, or both.

In another aspect is provided methods for treating hearing losscomprising administering to a subject in need thereof, an effectiveamount of a population of the proliferative postmitotic hair cells ofthe ear disclosed herein. In some embodiments, the proliferative haircells are modified to overexpress CDK1 and CCNB1, or equivalentsthereof. In other embodiments, the proliferative hair cells are modifiedto overexpress CDK4, CCND1, or both.

In another aspect is provided methods of treating hearing loss byadministering to a subject in need thereof, an effective amount of acomposition comprising proliferation and/or cell cycle reentry factors(e.g., CDK1, CCNB1 and AURKB or equivalents thereof).

In some aspects, a proliferative hair cell (e.g., hair cell of the innerear) of the present disclosure can be used to treat a subject in needthereof. In some embodiments, the proliferative hair cell can beadministered to the subject in need thereof, where administration intothe subject of the proliferative hair cell, treats hearing loss in thesubject. Thus, in some embodiments, a method of treating hearing lossinvolves administering to a subject in need thereof a population of haircells induced to proliferate and/or reenter the cell cycle. In otherembodiments, a method of treating hearing loss involves administering tothe subject in need thereof an effective amount of a compositioncomprising CDK1, CCNB1 and AURKB or equivalents thereof. In otherembodiments, a method of treating hearing loss involves administering tothe subject in need thereof an effective amount of a compositioncomprising CDK1, CDK4, CCNB1, CCND1, or combinations and/or equivalentsthereof.

Subjects in need of or suitable for treatment using the compositions,cells and methods of the present disclosure include, but are not limitedto, individuals having hearing loss caused by head trauma, viruses ordisease, exposure to loud noises, autoimmune inner ear diseases,hereditary disorders, aging (presbycusis), malformation of the innerear, and Meniere's disease.

Skeletal Muscle

In one aspect is provided methods for increasing muscle mass comprisingadministering to a subject in need thereof an effective amount of acomposition that increases the expression of CDK1 and CCNB1, orequivalents of each thereof. In some embodiments, the methods furthercomprise administering an effective amount of a composition thatincreases the expression of CDK4, CCND1, or both.

In another aspect is provided methods for increasing muscle masscomprising administering to a subject in need thereof, an effectiveamount of a population of the proliferative postmitotic skeletal musclecells disclosed herein. In some embodiments, the proliferative skeletalmuscle cells are modified to overexpress CDK1 and CCNB1, or equivalentsthereof. In other embodiments, the skeletal muscle cells are modified tooverexpress CDK4, CCND1, or both.

In another aspect is provided methods of increasing muscle mass byadministering to a subject in need thereof, an effective amount of acomposition comprising proliferation and/or cell cycle reentry factors(e.g., CDK1, CCNB1 and AURKB or equivalents thereof).

In some aspects, a proliferative skeletal muscle cell of the presentdisclosure can be used to treat a subject in need thereof. In someembodiments, the proliferative skeletal muscle cell can be administeredto the subject in need thereof, where administration into the subject ofthe skeletal muscle cell, builds muscle mass in the subject byincreasing the number of skeletal muscle cells. Thus, in someembodiments, a method of building muscle mass involves administering toa subject in need thereof a population of skeletal muscle cells inducedto proliferate and/or reenter the cell cycle. In other embodiments, amethod of building muscle mass involves administering to the subject inneed thereof an effective amount of a composition comprising CDK1, CCNB1and AURKB or equivalents thereof. In other embodiments, a method ofbuilding muscle mass involves administering to the subject in needthereof an effective amount of a composition comprising CDK1, CDK4,CCNB1, CCND1, or combinations and/or equivalents thereof.

Subjects in need of or suitable for use of the compositions, cells andmethods of the present disclosure include, but are not limited to,individuals having a skeletal muscle disease such as muscular dystrophy,cerebral palsy, amyotrophic lateral sclerosis, and myasthenia gravis orindividuals that desire an increase in muscle mass, such as weightlifters and bodybuilders.

Unless stated otherwise, the abbreviations used throughout thespecification have the following meanings:

-   -   Aa=amino acid(s)    -   Bp=base pair(s)    -   H=hour(s)    -   G=gram(s)    -   Kb=kilobase    -   kDa=kiloDalton    -   Kg=kilogram    -   L=liter    -   LC=liquid chromatography    -   Mg=milligram    -   Min=minute    -   mL=milliliter    -   mM=millimolar    -   nM=nanomolar    -   nT=nucleotide(s)    -   pM=picomolar    -   s.d.=standard deviation    -   μCi=microcurie    -   Mg=microgram    -   μL=microliter    -   μM=micromolar    -   Mm=micrometer    -   ° C.=degree Celsius

These one-letter symbols have the following meaning when representingamino acids:

-   -   A=Alanine    -   R=Arginine    -   N=Asparagine    -   D=Aspartic acid    -   C=Cysteine    -   E=Glutamic acid    -   Q=Glutamine    -   G=Glycine    -   H=Histidine    -   I=Isoleucine    -   L=Leucine    -   K=Lysine    -   M=Methionine    -   F=Phenylalanine    -   P=Proline    -   S=Serine    -   T=Threonine    -   W=Tryptophan    -   Y=Tyrosine    -   V=Valine

EXAMPLES

The following examples are intended to further illustrate certainembodiments of the disclosure. The examples are put forth so as toprovide one of ordinary skill in the art and are not intended to limitits scope.

Example 1. Induction of Proliferation in Non-ProliferativeCardiomyocytes

Cardiomyocyte Cell Culture:

Mouse cardiomyocytes were isolated from neonatal (newborn P0) and adult(6 week-old) as described in Ieda et al. (2009) Dev. Cell 16(2):233-244. Cardiomyocytes were cultured in DMEM/M199 medium containing 10%FBS.

Microarray Protocol:

Mouse genome-wide gene expression analyses were performed usingAffymetrix Mouse Gene 1.0 ST Array. RNA was extracted from neonatal(newborn P0) and adult (6 week-old) cardiomyocytes using Trizol(Invitrogen). Microarray analyses were performed in triplicate fromindependent biologic samples, according to the standard AffymetrixGenechip protocol. Data were analyzed using the Affymetrix Power Tool(APT, version 1.8.5). Differential gene expression was defined using thestatistics/threshold combination.

Overexpression:

Adenoviruses were constructed according to Mohamed et al., CardiovascRes. 2014 Jul. 1; 103(1):47-59. Briefly, the coding regions of EGFP,CDK1, CCNB1 and AURKB were amplified by PCR and subcloned intopENTR™/SD/D-TOPO shuttle vector. Adenoviruses were generated byhomologues recombination between the pENTR™/SD/D-TOPO shuttle vector andpAd/CMVN5-DEST vector (Life Technologies) using the Gateway® cloningsystem following the manufacturer's recommended methods. The adenovirusparticles were produced by transfecting the adenovirus encoding plasmidinto HEK293 cells and purified using Cesium Chloride column andtitrated. To infect the cardiomyocytes 25 multiplicity of infection(MOI) of the adenovirus were incubated for 24 hours and then the mediumwas replaced with fresh medium.

Immunocytochemistry:

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature,permeabilized with Saponin, blocked, and incubated with primaryantibodies against Troponin T (cTnT) (Thermo Scientific), phosphohistone H3 (PHH3) (Life Technologies), then with secondary antibodiesconjugated with Alexa 488 (green) or 594 (red) (Molecular Probes), and4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

Histology:

For Masson's trichrome staining, hearts were harvested 6 weeks followingmyocardial infarction and embedded in paraffin and transverselysectioned. Deparaffinized sections were dipped sequentially intoWeigert's iron hematoxylin working solution for 10 min, Biebrichscarlet-acid fuchsin solution for 15 min,phosphomolybdic-phosphotungstic acid solution for 15 min, aniline bluesolution 5-10 min, and 1% acetic acid solution for 2-5 min. Forimmunohistochemical studies in cell-injected hearts, hearts were fixedin 0.4% paraformaldehyde overnight, embedded in OCT compound, and frozenin liquid nitrogen (Ieda et al., 2010; Ieda et al., 2009). Hearts werecut vertically in 7-μm sections to show both ventricles. Sections werestained with primary antibodies against PHH3 (S10) and Troponin T(cTnT), with secondary antibodies conjugated with Alexa 488 or 594, andDAPI.

Proliferation and Cell Count Assays:

Two month old hiPSC-derived cardiomyocytes overexpressing CDK1, CCNB1and AURKB or a control adenoviral vector were grown in culture. EdU(5-ethynyl-2′-deoxyuridine; 10 μM) was administered to culture mediumfor 30 minutes. The incorporation of EdU was detected using the Click-iTEdU Cell Proliferation Assay Kit (Invitrogen Inc.) and analysed bymicroscopy.

The number of cardiomyocyte proliferating nuclei for two month oldhiPSC-derived cardiomyocytes transduced with a control or CDK1, CCNB1and AURKB adenovirus were quantified by assessing total nuclei countsper image using immunofluorescence for DAPI-positive cells.

Total cell numbers in populations of hiPSC-derived cardiomyocytestransduced with either a control or CDK1, CCNB1 and AURKB for 72 hourswere determined by FACS analysis. Cells were harvested from cultureddishes and analyzed on a FACS Calibur (BD Biosciences) with FlowJosoftware.

Cell number and viability were also analyzed using Cell Titre Glo(Promega, Madison, Wis., USA). Cell Titre Glo is a highly sensitiveindicator for cell number and viability through the measurement of ATPmetabolism, without the need to detach the cells from the culture plateas is required in FACS. Following incubation of control cells or cellstransduced with CDK1, CCNB1 and AURKB with the Cell Titre Glo reagent,luminescent intensities of the wells containing Cell Titre Glo wererecorded, with the intensity being proportional to the number of viablemetabolizing cells in the well.

Time Lapse Imaging:

Cardiomyocytes transduced with adenovirus to overexpress CDK1, CCNB1 andAURKB were plated into 24-well plates (10,000 cells per well) inquadruplicate and analyzed for cell proliferation using the IncuCyteimager (Essen BioSciences, Ann Arbor, Mich., USA). The plates werescanned in the IncuCyte at 1-hour intervals for 4 days. The data wereanalyzed with the IncuCyte software.

Animal Studies:

CDK1AF, CCNB1, and AURKB or GFP control adenovirus were injected intoC57bl6/N hearts at the site of injury following myocardial infarction(MI) as described in Qian et al., (2014) Nature 485(7400):593-8, Saxenaet al. (2008) Circulation 117: 2224-2231. Hearts were harvested fivedays post infarct to evaluate cell proliferation or six weeks postinfarct to evaluate cardiac function and for histology.

Echocardiography:

Systolic function was assessed with the use of 2-dimensionalmeasurements to measure ejection fraction (EF) in mice infected withCDK1AF, CCNB1 and AURKB or GFP control adenovirus. Mice wereanesthetized with 1.75% isoflurane. Core temperature was maintained at37-38° C., and scans were performed in a random-blind fashion. Percentejection fraction (% EF) and decline in EF over time were measured. Eachmouse underwent three separate scans on day 3-, 10-, 24- and 40-dayspost infarction.

Results

Cell Cycle Genes are Differentially Regulated Between Neonatal and AdultCardiomyocytes

To select potential factors that induce cardiac cell proliferationand/or cell cycle reentry, microarray analysis was performed to identifycell cycle genes differentially expressed between neonatal (newborn P0)and adult (6 week-old) mouse cardiomyocytes. Representative cell cyclegenes differentially expressed between proliferative (neo-natal) andnon-proliferative (adult) cardiomyocytes are shown in FIG. 1.

CDK1, CCNB1 and AURKB Promote Cardiac Cell Proliferation in Mouse andHuman Cardiomyocytes

The top fifteen down-regulated cell cycle genes in adult cardiomyocyteswere tested to determine whether overexpression of any one gene couldincrease proliferation and/or cell cycle reentry detected by EdUincorporation. Cardiomyocytes overexpressing AURKB (Aurora), CCNB1,CDC5, CDK1, CDK2, CDK3, CENPA, COPS5, CUL3, CYK4, and PRC1 showedincreased EdU incorporation (FIG. 2, panel A). On the other hand,overexpression of ANLN, CDH6 and WISP1 showed a decrease in EdUincorporation as compared to the control (FIG. 2, panel A). Theproliferative cells (EdU⁺) also stained positive for the cardiac cellmarker cardiac troponin T (cTnT) (FIG. 2, panel B).

It was next tested whether three factors (CDK1, CCNB1 and AURKB) used incombination are sufficient to induce proliferation of non-proliferativehiPSC-derived cardiomyocytes. CDK1, CCNB1 and AURKB were introduced intohiPSC-derived cardiomyocytes by adenovirus. Phospho histone H3(S10)-positive cells were not observed in control cells. In contrast,cells overexpressing CDK1, CCNB1 and AURKB for 72 hours showed anincrease in phospho histone H3 expression (FIG. 3). Similarly, when aconstitutively active CDK1 (CDK1AF) as well as CCNB1 and AURKB wereintroduced into cardiomyocytes with no proliferative capacity, isolatedfrom 7 day-old mice there was an increase in phospho histone H3expression as compared to cells transduced with a control adenovirus(FIG. 7).

Overexpression of CDK1, CCNB1 and AURKB Increase Cardiomyocyte CellNumber

Next, quantification of cell number in populations of cardiomyocytesoverexpressing CDK1, CCNB1 and AURKB was performed. Three independentmethods were used. Quantification of proliferating cardiomyocyte nucleishowed that there is doubling in the number of nuclei in response tooverexpression of CDK1, CCNB1 and AURKB for 72 hours (FIG. 4, panel A).In addition, 30% of the total nuclei stained positive for PHH3. FACSanalysis showed that the cell number in populations of cardiomyocytesoverexpressing CDK1, CCNB1 and AURKB increased by 2-fold compared to thecontrol (FIG. 4, panel B). To test for cell number and cell viabilitywithout the need to detach cells from the culture dish, as required byFACS, ATP Glo assays were performed. Using this method, a 2-foldincrease in the cell number following overexpression of CDK1, CCNB1 andAURKB for 72 hours was observed—similar to that reported in FACSanalysis (FIG. 4, panel C).

Time lapse imaging was performed to further assess the induction ofcardiomyocyte proliferation by overexpression of CDK1, CCNB1 and AURKB.Images were collected hourly over the course of 4 days. Cell divisionswere observed in both mouse and human cardiomyocytes overexpressingCDK1, CCNB1 and AURKB (FIGS. 5 and 6).

Together these data demonstrated that non-proliferative adultcardiomyocytes can be induced to proliferate and/or reenter the cellcycle by overexpressing cell cycle genes such as CDK1, CCNB1 and AURKB.

CDK1, CCNB1 and AURKB Improve Cardiac Function and Structure afterMyocardial Infarction

To determine whether inducing cardiomyocyte proliferation withoverexpression of CDK1, CCNB1 and AURKB is beneficial after myocardialinjury CDK1AF, CCNB1 and AURKB adenovirus or control GFP adenovirus wasinjected into mice at the site of injury following myocardialinfarction. At 5 days post-infarct, hearts showed increasedproliferation at the site of injury in CDK1AF, CCNB1 and AURKB injectedhearts as compared to the control GFP injected heart (FIG. 8, panel A).In addition, at 6 weeks post-infarct, three out of five hearts injectedwith CDK1AF, CCNB1 and AURKB demonstrated a significant amount ofcardiomyocytes within the infarct area compared to the GFP control (FIG.8, panel B).

To assess cardiac function, animals were injected with either CDK1AF,CCNB1 and AURKB or GFP control adenovirus. The animals were followed byechocardiography to assess the heart function at 3-, 10-, 24- and40-days post-infarct. Only one out of five controls survived. On theother hand, five out of seven mice injected with CDK1AF, CCNB1 and AURKBsurvived (FIG. 9, panel A). In addition, four out of five mice injectedwith CDK1AF, CCNB1 and AURKB showed less deterioration in cardiacfunction overtime compared to the only survivor control (FIG. 9, panelB).

These in vivo data demonstrated that adult cardiomyocytes induced toproliferate and/or reenter the cell cycle can improve cardiac functionand structure after myocardial infarction.

Example 2. Induction of Proliferation in Postmitotic HumanCardiomyocytes

To determine an optimal cocktail of factors for inducing cellproliferation and/or cell cycle reentry of human postmitoticcardiomyocytes, combinations of factors were screened. Human embryonicstem cell-derived cardiomyocytes were generated as previously describedLian, X. et al. (2013) Nat Protoc 8(1): 162-175. Cells were treated withcombinations of CDK1AF, CDK4, CCNB1, CCND1, AURKB or GFP controladenovirus, with or without combinations of TGF-βi (SB431542, “SB”), aCDK activator (MK1775, “MK”), and IGF1. Analysis was performed toidentify cocktails of factors that increased the number of cellsundergoing mitosis as assessed by increased phospho-histone H3 stainingat 4-, 8-, and 12-days after treatment with factor cocktails. FIG. 10,panel A. Analysis was also performed to identify which cocktails offactors had the highest cell survival as assessed by the increase intotal number of nuclei. FIG. 10, panel B.

These data demonstrated that the combination of CDK1AF, CDK4, CCNB1, andCCND1 was the cocktail of factors screened that resulted in bestsurvival and increase in proliferative postmitotic cardiomyocytes. Itwas also observed that inhibition of TGFβ signaling using SB431542enhanced survival. In addition, activation of CDK1 with small moleculeMK1775 could replace CDK1 and CCNB1 from the cocktail.

Example 3. Cocktails Target Different Stages of the Cell Cycle

To better understand the mechanisms of action by which the differentcocktails of factors promote cell proliferation and/or cell cyclereentry of postmitotic cells, cell cycle behavior of cells wasvisualized using Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI)technology. Zielke et al. (2015) WIREs Dev Biol 4:469-487. Using thissystem, cells in M/G1 phase fluoresce red (mCherry), cells in S/G2fluoresce green (GFP), and cell in S fluoresce red and green. Comparisonof the cocktail combination of CDK1, CCNB1, and AURKB (panel A) with thecocktails of CDK4 and CCND (panel B) and CDK1, CCNB, CDK4, and CCND(panel C) showed that the combination of CDK1, CCNB1, and AURKB acted asa G2 cocktail and enhanced the number of cells in G2, but not G1 or S.The combination of CDK4 and CCND1 acted as a G1 cocktail and enhancedthe number of cells in G1 and S but not G2. On the other hand, thecombination of CDK1, CCNB1, CDK4 and CCND1 induced a balanceddistribution of G1/G2 cells. It is believed that the balanceddistribution aids in cell survival.

Example 4. CDK1/CCNB1/CDK4/CCND1 Cocktail Improves Cardiac Functionafter Myocardial Infarction

To determine whether the G1/G2 cocktail of CDK1/CCNB1/CDK4/CCND1 couldimprove cardiac function following a myocardial infarction, in vivostudies were performed. The animal protocol for surgery was approved bythe University of California, San Francisco Institutional Animal Careand Use Committee. All surgeries were performed as previously described.Qian, L., et al. (2012) Nature 485:593-598. Briefly, mice wereanaesthetized with 2.4% isoflurane/97.6% oxygen and placed in a supineposition on a heating pad (37° C.). Animals were intubated with a 19 Gstump needle and ventilated with room air using a MiniVent Type 845mouse ventilator (Hugo Sachs Elektronik-Harvard Apparatus; strokevolume, 250 μl; respiratory rate, 120 breaths per minute). Myocardialinfarction (MI) was induced by permanent ligation of the left anteriordescending (LAD) artery with a 7-0 prolene suture as described in Qian,L., et al. (2012) Nature 485:593-598. Sham-operated animals served assurgical controls and were subjected to the same procedures as theexperimental animals with the exception that the LAD was not ligated. ATthe time of MI, the animals received an injection of the G1/G2 cocktailinto the myocardium.

Serial echocardiography was conducted before MI and 1, 2, 4, 8 and 12weeks after MI to assess the cardiac function. Echocardiography wasperformed with the Vevo 770 High-Resolution Micro-Imaging System(VisualSonics) with a 15-MHz linear-array ultrasound transducer. Theleft ventricle was assessed in both parasternal long-axis and short-axisviews at a frame rate of 120 Hz. End-systole or end-diastole weredefined as the phases in which the left ventricle appeared the smallestand largest, respectively, and used for ejection-fraction measurements.To calculate the shortening fraction, left-ventricular end-systolic andend-diastolic diameters were measured from the left ventricular M-modetracing with a sweep speed of 50 mm/s at the papillary muscle. B-modewas used for two-dimensional measurements of end-systolic andend-diastolic dimensions.

Standard Masson's Trichrome staining was performed on hearts 12 weekspost-viral delivery and coronary artery ligation. To determine scarsize, ImagePro software was used to measure the scar area (blue) andhealthy area (red) on transverse sections spanning four levels withinthe left ventricle of an MI heart.

Results show that treatment with the G1/G2 cocktail significantlyenhanced cardiac function compared to sham treated animal, as reflectedby changes in the ejection fraction (EF) assessed by echocardiography(FIG. 12). The improved function occurred as early as 1 week after MI.Histological analyses were performed to quantify the scar size anddetect the presence of muscle within the infarct area of treated hearts.Consistent with the in vivo imaging observations, it was found in heartsisolated from animals treated with G1/G2 cocktail, thicker bands ofmyocytes were observed within the infarct zone (FIG. 13, panel A). Inaddition, scar size was significantly reduced in treated animals (FIG.13, panel B).

To confirm that this remuscularization was due to cardiomyocyteproliferation in vivo, Mosaic Analysis of Dual Marker (MADM) mice wereused to perform lineage tracing. In cells isolated from these mice, whena cell divides it will rise to a green and a red cell and if there is nodivision it will stay yellow and, therefore, distinguish betweendividing and non-dividing cells. It was found that the remuscularizationaround the infarct area was due, at least in part, to generation of newcardiomyocytes through proliferation of endogenous cells (FIG. 14).

Example 5. Induction of Proliferation in Postmitotic Neurons

To determine whether the G1/G2 cocktail could also induce cellproliferation and/or cell cycle reentry of postmitotic neurons, primarycultures of cortical neurons were prepared from rat neonates (P16-18) asreported in Arrasate, M. et al. (2005) Proc Natl Acad Sci USA102(10):3840-3845. Brain cortices were dissected, treated with papainand then trypsin inhibitor, and gently triturated to dissociate singleneurons in Optimem_glucose medium. Cells (approximately 150,000) wereplated in each well of a 96-well tissue culture plate. Two hours later,the plating medium was replaced by growth medium with serum. Cells werelabeled with pGW1-GFP as a morphology marker and neuronal specificpromoter MAP2-DsRed to indicate that these cells are mature neurons.Time lapse microscopy was performed to identify dividing cells. As shownin FIG. 15, the G1/G2 cocktail induced cell division of postmitoticneurons. In addition, it was determined that most divisions occurredbetween 60-90 hours post-infection with the G1/G2 cocktail, while somedivisions occurred as late as 120 hours post-infection. FIG. 16.

Example 6. Induction of Proliferation in Postmitotic Pancreatic BetaCells

To determine whether the G1/G2 cocktail can induce cell proliferationand/or cell cycle reentry of postmitotic pancreatic β-cells, β-cells arepurified from other non-β-cell subpopulations, for example, by themethods described in Clardy et al. (2015) Scientific Reports 5:13681.Purified β-cells are plated into a tissue culture plate and treated withthe G1/G2 adenoviral cocktail or sham adenoviral control. β-cellstreated with the G1/G2 cocktail show an increase in cell divisions at48-, 72- and 96-hours post-infection. β-cells treated with the shamcocktail show no increase in cell division.

Example 7. Induction of Proliferation in Postmitotic Hair Cells

To determine whether the G1/G2 cocktail could also induce cellproliferation and/or cell cycle reentry of postmitotic hair cells of theinner ear, inner ear cells are derived in vitro from induced pluripotentstem cells using methods similar to those described in Ronaghi M et al.(2014) Stem Cells Dev 23(11)1275-1284. Differentiated inner ear cellsare plated into a tissue culture plate and treated with the G1/G2adenoviral cocktail or sham adenoviral control. Cells treated with theG1/G2 cocktail show an increase in cell divisions at 48-, 72- and96-hours post-infection. Cells treated with the sham cocktail show noincrease in cell division.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

In addition, where the features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup members of the Markush group.

All publications, patent applications, patents and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Human CDK1 Gene ID: 983mRNA: NM_001786 (transcript variant 1), CDS (143..1036) SEQ. ID. NO. 1   1 agcgcggtga gtttgaaact gctcgcactt ggcttcaaag ctggctcttg gaaattgagc  61 ggagagcgac gcggttgttg tagctgccgc tgcggccgcc gcggaataat aagccgggat 121 ctaccatacc cattgactaa ctatggaaga ttataccaaa atagagaaaa ttggagaagg 181 tacctatgga gttgtgtata agggtagaca caaaactaca ggtcaagtgg tagccatgaa 241 aaaaatcaga ctagaaagtg aagaggaagg ggttcctagt actgcaattc gggaaatttc 301 tctattaaag gaacttcgtc atccaaatat agtcagtctt caggatgtgc ttatgcagga 361 ttccaggtta tatctcatct ttgagtttct ttccatggat ctgaagaaat acttggattc 421 tatccctcct ggtcagtaca tggattcttc acttgttaag agttatttat accaaatcct 481 acaggggatt gtgttttgtc actctagaag agttcttcac agagacttaa aacctcaaaa 541 tctcttgatt gatgacaaag gaacaattaa actggctgat tttggccttg ccagagcttt 601 tggaatacct atcagagtat atacacatga ggtagtaaca ctctggtaca gatctccaga 661 agtattgctg gggtcagctc gttactcaac tccagttgac atttggagta taggcaccat 721 atttgctgaa ctagcaacta agaaaccact tttccatggg gattcagaaa ttgatcaact 781 cttcaggatt ttcagagctt tgggcactcc caataatgaa gtgtggccag aagtggaatc 841 tttacaggac tataagaata catttcccaa atggaaacca ggaagcctag catcccatgt 901 caaaaacttg gatgaaaatg gcttggattt gctctcgaaa atgttaatct atgatccagc 961 caaacgaatt tctggcaaaa tggcactgaa tcatccatat tttaatgatt tggacaatca1021 gattaagaag atgtagcttt ctgacaaaaa gtttccatat gttatatcaa cagatagttg1081 tgtttttatt gttaactctt gtctattttt gtcttatata tatttctttg ttatcaaact1141 tcagctgtac ttcgtcttct aatttcaaaa atataactta aaaatgtaaa tattctatat1201 gaatttaaat ataattctgt aaatgtgtgt aggtctcact gtaacaacta tttgttacta1261 taataaaact ataatattga tgtcaggaat caggaaaaaa tttgagttgg cttaaatcat1321 ctcagtcctt atggcagttt tattttcctg tagttggaac tactaaaatt taggaaaatg1381 ctaagttcaa gtttcgtaat gctttgaagt atttttatgc tctgaatgtt taaatgttct1441 catcagtttc ttgccatgtt gttaactata caacctggct aaagatgaat atttttctac1501 tggtatttta attttagacc taaatgttta agcattcgga atgagaaaac tatacagatt1561 tgagaaatga tgctaaattt ataggagttt tcagtaactt aaaaagctaa catgagagca1621 tgccaaaatt tgctaagtct tacaaagatc aagggctgtc cgcaacaggg aagaacagtt1681 ttgaaaattt atgaactatc ttatttttag gtaggttttg aaagcttttt gtctaagtga1741 attcttatgc cttggtcaga gtaataactg aaggagttgc ttatcttggc tttcgagtct1801 gagtttaaaa ctacacattt tgacatagtg tttattagca gccatctaaa aaggctctaa1861 tgtatattta actaaaatta ctagctttgg gaattaaact gtttaacaaa taaaaaaaaa1921 aaa Human CDK1 SEQ. ID. NO. 2MEDYTKIEKIGEGTYGVVYKGRHKTTGQVVAMKKIRLESEEEGVPSTAIREISLLKELRHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYMDSSLVKSYLYQILQGIVFCHSRRVLHRDLKPQNLLIDDKGTIKLADFGLARAFGIPIRVYTHEVVTLWYRSPEVLLGSARYSTPVDIWSIGTIFAELATKKPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHVKNLDENGLDLLSKMLIYDPAKRISGKM ALNHPYFNDLDNQIKKMHuman Cyclin B1 (CCNB1) GeneID: 891 mRNA: NM_031966.3, CDS (254..1555)SEQ. ID. NO. 3    1cgaacgcctt cgcgcgatcg ccctggaaac gcattctctg cgaccggcag ccgccaatgg   61gaagggagtg agtgccacga acaggccaat aaggagggag cagtgcgggg tttaaatctg  121aggctaggct ggctcttctc ggcgtgctgc ggcggaacgg ctgttggttt ctgctgggtg  181taggtccttg gctggtcggg cctccggtgt tctgcttctc cccgctgagc tgctgcctgg  241tgaagaggaa gccatggcgc tccgagtcac caggaactcg aaaattaatg ctgaaaataa  301ggcgaagatc aacatggcag gcgcaaagcg cgttcctacg gcccctgctg caacctccaa  361gcccggactg aggccaagaa cagctcttgg ggacattggt aacaaagtca gtgaacaact  421gcaggccaaa atgcctatga agaaggaagc aaaaccttca gctactggaa aagtcattga  481taaaaaacta ccaaaacctc ttgaaaaggt acctatgctg gtgccagtgc cagtgtctga  541gccagtgcca gagccagaac ctgagccaga acctgagcct gttaaagaag aaaaactttc  601gcctgagcct attttggttg atactgcctc tccaagccca atggaaacat ctggatgtgc  661ccctgcagaa gaagacctgt gtcaggcttt ctctgatgta attcttgcag taaatgatgt  721ggatgcagaa gatggagctg atccaaacct ttgtagtgaa tatgtgaaag atatttatgc  781ttatctgaga caacttgagg aagagcaagc agtcagacca aaatacctac tgggtcggga  841agtcactgga aacatgagag ccatcctaat tgactggcta gtacaggttc aaatgaaatt  901caggttgttg caggagacca tgtacatgac tgtctccatt attgatcggt tcatgcagaa  961taattgtgtg cccaagaaga tgctgcagct ggttggtgtc actgccatgt ttattgcaag 1021caaatatgaa gaaatgtacc ctccagaaat tggtgacttt gcttttgtga ctgacaacac 1081ttatactaag caccaaatca gacagatgga aatgaagatt ctaagagctt taaactttgg 1141tctgggtcgg cctctacctt tgcacttcct tcggagagca tctaagattg gagaggttga 1201tgtcgagcaa catactttgg ccaaatacct gatggaacta actatgttgg actatgacat 1261ggtgcacttt cctccttctc aaattgcagc aggagctttt tgcttagcac tgaaaattct 1321ggataatggt gaatggacac caactctaca acattacctg tcatatactg aagaatctct 1381tcttccagtt atgcagcacc tggctaagaa tgtagtcatg gtaaatcaag gacttacaaa 1441gcacatgact gtcaagaaca agtatgccac atcgaagcat gctaagatca gcactctacc 1501acagctgaat tctgcactag ttcaagattt agccaaggct gtggcaaagg tgtaacttgt 1561aaacttgagt tggagtacta tatttacaaa taaaattggc accatgtgcc atctgtacat 1621attactgttg catttacttt taataaagct tgtggcccct tttacttttt tatagcttaa 1681ctaatttgaa tgtggttact tcctactgta gggtagcgga aaagttgtct taaaaggtat 1741ggtggggata tttttaaaaa ctccttttgg tttacctggg gatccaattg atgtatatgt 1801ttatatactg ggttcttgtt ttatatacct ggcttttact ttattaatat gagttactga 1861aggtgatgga ggtatttgaa aattttactt ccataggaca tactgcatgt aagccaagtc 1921atggagaatc tgctgcatag ctctatttta aagtaaaagt ctaccaccga atccctagtc 1981cccctgtttt ctgtttcttc ttgtgattgc tgccataatt ctaagttatt tacttttacc 2041actatttaag ttatcaactt tagctagtat cttcaaactt tcactttgaa aaatgagaat 2101tttatattct aagccagttt tcattttggt tttgtgtttt ggttaataaa acaatactca 2161aatacaaaaa aaaaaaa Human Cyclin B1 (CCNB1) SEQ. ID. NO. 4MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASPSPMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKVHuman Aurora kinase B (AURKB) GeneID:9212mRNA: NM_004217.3 (transcript variant 1), CDS (123..1157) SEQ. ID. NO. 5   1 cggggcggga gatttgaaaa gtccttggcc agggcgcggc gtggcagatt cagttgtttg  61 cgggcggccg ggagagtagc agtgccttgg accccagctc tcctccccct ttctctctaa 121 ggatggccca gaaggagaac tcctacccct ggccctacgg ccgacagacg gctccatctg 181 gcctgagcac cctgccccag cgagtcctcc ggaaagagcc tgtcacccca tctgcacttg 241 tcctcatgag ccgctccaat gtccagccca cagctgcccc tggccagaag gtgatggaga 301 atagcagtgg gacacccgac atcttaacgc ggcacttcac aattgatgac tttgagattg 361 ggcgtcctct gggcaaaggc aagtttggaa acgtgtactt ggctcgggag aagaaaagcc 421 atttcatcgt ggcgctcaag gtcctcttca agtcccagat agagaaggag ggcgtggagc 481 atcagctgcg cagagagatc gaaatccagg cccacctgca ccatcccaac atcctgcgtc 541 tctacaacta tttttatgac cggaggagga tctacttgat tctagagtat gccccccgcg 601 gggagctcta caaggagctg cagaagagct gcacatttga cgagcagcga acagccacga 661 tcatggagga gttggcagat gctctaatgt actgccatgg gaagaaggtg attcacagag 721 acataaagcc agaaaatctg ctcttagggc tcaagggaga gctgaagatt gctgacttcg 781 gctggtctgt gcatgcgccc tccctgagga ggaagacaat gtgtggcacc ctggactacc 841 tgcccccaga gatgattgag gggcgcatgc acaatgagaa ggtggatctg tggtgcattg 901 gagtgctttg ctatgagctg ctggtgggga acccaccctt tgagagtgca tcacacaacg 961 agacctatcg ccgcatcgtc aaggtggacc taaagttccc cgcttccgtg cccatgggag1021 cccaggacct catctccaaa ctgctcaggc ataacccctc ggaacggctg cccctggccc1081 aggtctcagc ccacccttgg gtccgggcca actctcggag ggtgctgcct ccctctgccc1141 ttcaatctgt cgcctgatgg tccctgtcat tcactcgggt gcgtgtgttt gtatgtctgt1201 gtatgtatag gggaaagaag ggatccctaa ctgttccctt atctgttttc tacctcctcc1261 tttgtttaat aaaggctgaa gctttttgta ctcatgaaaa aaaaaaaaaa aaaaHuman Aurora kinase B (AURKB) SEQ. ID. NO. 6MAQKENSYPWPYGRQTAPSGLSTLPQRVLRKEPVTPSALVLMSRSNVQPTAAPGQKVMENSSGTPDILTRHFTIDDFEIGRPLGKGKFGNVYLAREKKSHFIVALKVLFKSQIEKEGVEHQLRREIEIQAHLHHPNILRLYNYFYDRRRIYLILEYAPRGELYKELQKSCTFDEQRTATIMEELADALMYCHGKKVIHRDIKPENLLLGLKGELKIADFGWSVHAPSLRRKTMCGTLDYLPPEMIEGRMHNEKVDLWCIGVLCYELLVGNPPFESASHNETYRRIVKVDLKFPASVPMGAQDLISKLLRHNPSERLPLAQVSAHPWVRANSR RVLPPSALQSVAMouse CDK1 GeneID: 12534 mRNA: NM_007659.3, CDS (104..997)SEQ. ID. NO. 7    1actcggcctc taagctcctg gagttgctgc gtccgcgcag tccggaactg cggtgtggcc   61ccagccggga cagagagggt ccgtcgtaac ctgttgagta actatggaag actatatcaa  121aatagagaaa attggagaag gtacttacgg tgtggtgtat aagggtagac acagagtcac  181tggccagata gtggccatga agaagatcag acttgaaagc gaggaagaag gagtgcccag  241tactgcaatt cgggaaatct ctctattaaa agaacttcga catccaaata tagtcagcct  301gcaggatgtg ctcatgcagg actccaggct gtatctcatc tttgagttcc tgtccatgga  361cctcaagaag tacctggact ccatccctcc tgggcagttc atggattctt cactcgttaa  421gagttactta caccaaatcc tccagggaat tgtgttttgc cactcccggc gagttcttca  481cagagacttg aaacctcaaa atctattgat tgatgacaaa ggaacaatca aactggctga  541tttcggcctt gccagagcgt ttggaatacc gatacgagtg tacacacacg aggtagtgac  601gctgtggtac cgatctccag aagtgttgct gggctcggct cgttactcca ctccggttga  661catctggagt atagggacca tatttgcaga actggccacc aagaagccgc ttttccacgg  721cgactcagag attgaccagc tcttcaggat cttcagagct ctgggcactc ctaacaacga  781agtgtggcca gaagtcgagt ccctgcagga ctacaagaac acctttccca agtggaagcc  841ggggagcctc gcatcccacg tcaagaacct ggacgagaac ggcttggatt tgctctcaaa  901aatgctagtc tatgatcctg ccaaacgaat ctctggcaaa atggccctga agcacccgta  961ctttgatgac ttggacaatc agattaagaa gatgtagccc tctggatgga tgtccctgtc 1021tgctggtcgt aggggaagat cgtgttgttt accgttggct ctcttcctgt cttgtatagt 1081tttctttgtt tgtaaactgt catctggact tttcttaatt tcctacgtat aacttaatta 1141acatgtaaat attattccat atgaatttaa atataattct gtatatgtgc agatgtcact 1201gtggtggctg ttaattacta taacacaagt gttaattact acaacataag acttgagtct 1261ccctagactt cccagcagcc attcctgcag ctcggagcac agttgaagga gctgagctca 1321ggcctcgtga tgctttcaag tgcctccgtg ttctggatat atatgattcc tggtcagttt 1381cttgccattt ggaaactaca acccacctac ggacagtgtt tttctacttg tgcttaagca 1441gttgggatga gaaggccaaa gacccgagga tgtctagagt aatgaccccc agatggaagt 1501gcaccaaagc tggctgggtt tcacagctag agatcagggg ctgtccagag caggacagct 1561tagaacattt atgaagactc cctatttttt ggtttgttgt aaagctgttg tctagttgga 1621ttcctgtgct ctgcatggtc agaggtaggt tagaggattt gccttggctt ctaaatccaa 1681tttgaaaact gcttaaaatc tcctgtcctc tcttagcagt gtctaaaaat gtccttgtcc 1741aaatatttag ctgagattcc tcactttgga aaaggagccg tatcgctgtg ctgcttagtg 1801taattcttag aagcagcctg acttatctgc tagcagtcaa agggatgcct gagacgactg 1861ctcctcttag aactaaaggc tgggatgctt aagtttgtct actgtttgga ggatctcggt 1921aagactgagc ccctgttcct gtggccacct cagtttacca gtacctcagc ctcagcctcc 1981tgcatttgct ggagtcaggg aaggtcccca gccctgagcc ctgacgctcc tgattgtaga 2041gactgtcagt tggaggtaaa acgttcattg aagtagtcag cagccacatg catttagggc 2101actccagtgt cagagaccat cctggaggtt tctaaccctg ccgctggcag tctactccca 2161agacagatca gttagagtgg tcagcaaaca ccaactgctg cagaaacctg tgttggtgtg 2221gttccctgct gctgctggga actgggccca agactagaga gcttggggct gcggttgatc 2281atgggttctg ttcctgcatt acaccttgaa atccaagcct tctaatatct cccttcggat 2341cataagttgt gaatttggtc ctccgccccc gccaggtttt ctatacttgg gtttgtcttg 2401ctgacatttt caagagtcct gactaagacg gtgattagtg tgacatgact tgagaactac 2461cgatttgaag cacacttgaa gttaacaaat tctctcatga ttatactttt aactattatt 2521aagattgctt gagctcaccc agatctctgt tgggaagtaa ctgggtaaca aaagccgttg 2581cactggtttg acagctaaca actgttggta ctttgtattc agaaggaatg aggtagcgat 2641tgaatggctg gggtgttgtt tccacagttt atacactaaa aatttgggta gccaggaggt 2701agtggcgcgc aatctttaat ctcagcagag gcagtcctat ctcttgagtt ggaggccagc 2761ctgcctgagt tccagaccag ccagggctac aggaagaagt cttaaaaaaa tattttttcc 2821ctgtggatgt aaacccatga gaatgactgc tgtatctatc Mouse CDK1Amino Acid Sequence SEQ. ID. NO. 8MEDYIKIEKIGEGTYGVVYKGRHRVTGQIVAMKKIRLESEEEGVPSTAIREISLLKELRHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQFMDSSLVKSYLHQILQGIVFCHSRRVLHRDLKPQNLLIDDKGTIKLADFGLARAFGIPIRVYTHEVVTLWYRSPEVLLGSARYSTPVDIWSIGTIFAELATKKPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHVKNLDENGLDLLSKMLVYDPAKRISGKM ALKHPYFDDLDNQIKKMMouse Cyclin B1 (CCNB1) GeneID: 268697 mRNA: NM_172301.3, CDS (85..1377)SEQ. ID. NO. 9    1ggaacggctg ttagtgttta gctgtggata gccagaggtt agggtgtctt ctcgaatcgg   61ggaacctctg attttggagg agccatggcg ctcagggtca ctaggaacac gaaaattaac  121gcagaaaata aggccaaggt cagtatggca ggcgccaagc gtgtgcctgt gacagttact  181gctgcttcca agcccgggct gagaccgaga actgctcttg gagacattgg taataaagtc  241agcgaagagc tacaggcaag agtgcctctg aaaagggaag caaaaacgct aggtactgga  301aaaggtactg ttaaagccct accaaaacct gtagagaagg tgcctgtgtg tgaaccagag  361gtggaacttg ctgagcctga gcctgaacct gaacttgaac atgttagaga agagaagctt  421tctcctgaac ctattttggt tgataatccc tctccaagcc cgatggaaac atctggatgt  481gcgcctgcag aagagtatct gtgtcaggct ttctctgatg taatccttgc agtgagtgac  541gtagacgcag atgatggggc tgacccaaac ctctgtagtg aatatgtgaa agatatctat  601gcttatctcc gacaactgga ggaagagcag tcagttagac caaaatacct acagggtcgt  661gaagtgactg gaaacatgag agctatcctc attgactggc taatacaggt tcagatgaaa  721tttaggctgc ttcaggagac catgtacatg actgtgtcca ttattgatcg gttcatgcag  781aacagttgtg tgcccaagaa gatgctacag ctggtcggtg taacggccat gtttattgca  841agcaaatatg aggagatgta ccctccagaa ataggtgact tcgcctttgt gactaacaac  901acgtacacta agcaccagat cagacagatg gagatgaaga ttctcagagt tctgaacttc  961agcctgggtc gccctctgcc tctgcacttc ctccgtagag catctaaagt cggagaggtt 1021gacgtcgagc agcacacttt ggccaaatac ctcatggagc tctccatgct ggactacgac 1081atggtgcatt ttgctccttc tcaaattgca gctggggctt tctgcttagc gctgaaaatt 1141cttgacaacg gtgaatggac accaactctg cagcactacc tatcctacag tgaagactcc 1201ctgcttcctg ttatgcagca cctggctaag aatgtagtca tggtgaactg tggcctcaca 1261aagcacatga ctgtcaagaa caagtatgca gcatctaagc atgctaagat cagcacgctg 1321gcacagctga actgtacact agttcagaat ttgtctaagg ccgtgacaaa ggcataactc 1381caatagactg ctacatctgc agatgcagtt ggcaccatgt gccgcctgta cataggatac 1441ctaccgtgtt tacttgctct tcaataaagg ttgtgacttc tcattttaca tagcttaact 1501catttgaatg ttgttgcttc tgagtttagg ctaacggaag ttgtcgaatt taggagtata 1561ttaaaaactg catctagttt taacagtgga tccaactaat gtatatatct gtagcctata 1621tgtctatata catccttcac tgtgtgtcct tatatcatca tgtcttctgc ctcactctag 1681tttaaactct aaatctacca gctagtcctt tgttccattt tccagtggtt gccaccttta 1741accactgtct cttggtttgt caactttcag atctgaaacc aagtatcttt tittatgtaa 1801ttatttattt gttcttaatt ggaaaatagg atgttcaaaa ttaaaggtgt gattaaaaa 1861gaatttgccc ccaagtctca ctatcaacag ataagggtgt attcttgtat atcctgtata 1921gatataatca tgcatatact cccaaggaga tatttttata tgggttcatt ttatcaacag 1981tattcctatc agcattcctt tcaatgccta tattgcattt cctagtgtga acaaactgtg 2041tgtaacatag tcattccctc ggtgggattc aagtgcattc tctcagtgcc ctccacagtg 2101ttcttaaatg atgtttaatg tcttgcttgg cttcattcat agtagctctt ccaggggtgt 2161gctttgaatt ctgacagcca gatgggtgtg gctgccacca taccaaggcg ccactcctgt 2221cttgtaatgc cacctggaaa agaatcctgt ctcatttgct gttttaattt atacatctga 2281tatcaagttg aataaaattt attggtggaa agcttt Mouse Cyclin B1 (CCNB1)Amino Acid Sequence SEQ. ID. NO. 10MALRVTRNTKINAENKAKVSMAGAKRVPVTVTAASKPGLRPRTALGDIGNKVSEELQARVPLKREAKTLGTGKGTVKALPKPVEKVPVCEPEVELAEPEPEPELEHVREEKLSPEPILVDNPSPSPMETSGCAPAEEYLCQAFSDVILAVSDVDADDGADPNLCSEYVKDIYAYLRQLEEEQSVRPKYLQGREVTGNMRAILIDWLIQVQMKFRLLQETMYMTVSIIDRFMQNSCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTNNTYTKHQIRQMEMKILRVLNFSLGRPLPLHFLRRASKVGEVDVEQHTLAKYLMELSMLDYDMVHFAPSQIAAGAFCLALKILDNGEWTPTLQHYLSYSEDSLLPVMQHLAKNVVMVNCGLTKHMTVKNKYAASKHAKISTLAQLNCTLVQNLSKAVTKAMouse Aurora kinase B (AURKB) GeneID:20877mRNA: NM_011496.1, CDS (319..1356) SEQ. ID. NO. 11    1ggagattcga aagcgtccgg gtcgcggggt aaaccggttc tccgtgtgcg agcgcctagt   61ggcgtaggct gcggctttgc ggggaactgc gggggctgca gtggtccacg gggctgatcg  121ggttccgttg ggcggatcca cgtgcccgct atccgcctgg aaggagaggt gcaggagtac  181ccccgacctt ggctgcgtgc tgactcgctt ccttctgccc gcccaggctt gcactccccg  241gggatctgcc tctgcatctc ttgccttcgc tgttgtttcc ctctctgtcc agctcccctc  301ccgctctcgc cctggagaat ggctcagaag gagaacgcct acccgtggcc ctacggctca  361aagacgtctc agtctggcct gaacacgttg tcccagagag tcctacggaa ggagcccgcc  421acgacatctg cgcttgctct cgtgaaccgg ttcaacagcc agtccacagc tgcccctggc  481cagaagttgg ctgagaacaa gagtcagggc tccactgcct cgcaaggatc ccagaacaag  541cagcctttca ctattgacaa ctttgagatt gggcgtcctt tgggcaaagg caaatttgga  601aacgtgtact tggctcggga gaagaagagc cgtttcatcg tggcactcaa gatcctcttc  661aagtctcaga ttgagaagga gggggtagag caccagcttc gccgagagat cgaaatccag  721gcgcacctga aacatcccaa catccttcaa ctctacaact acttctacga ccagcagagg  781atctacttaa tcctggaata cgcccctcgc ggggaactct acaaggaact gcagaagagt  841cggaccttcg atgagcagcg gactgccacg atcatggagg aactgtcaga tgccctgacc  901tactgccaca agaagaaggt aattcacaga gacataaagc cggagaacct gctgttaggt  961ctgcagggag aactgaagat tgcagacttt ggctggtcgg tgcatgcccc atccctgagg 1021aggaagacca tgtgcggcac gctggactat ctgcccccag agatgattga ggggcgcatg 1081cataatgaaa tggtagatct atggtgcatc ggggtgctct gctatgaact gatggtgggg 1141aacccaccct tcgagagccc tagccacagt gagacgtatc gtcggattgt caaggtggac 1201ctgaagttcc cctcttctgt gccttcgggc gcccaggacc tcatctccaa gctgctcaaa 1261cataacccct ggcaacggct gcccctggcg gaggttgcag ctcacccttg ggtccgggcc 1321aactcaagga gggttctgcc tccctctgcc ctttagcctg ctccttggtt ttttgtccct 1381gtcatttttc agtgttcttt gtatgtctgt gtatgtgttc tgagaagggg tgggaactgg 1441aaactattcc tagctccagt tctaggggat ctgatctctc ttctgacctc tacaggcaaa 1501attaggcacc cctgtggtgc acatatatgc acaccaaaca catgaagtta caaacaaaca 1561acaaacacac agatagtgct ggagagatgg ctcggtagtt aaaagcactg gctgctcttc 1621ccaggaacct agaactcaat tctagcacta catggtgctc acgaccactg tctgtaacac 1681ccagtcctgg ggaatctggg gccttcgagc ctctgcagac actaggcatg gatgtggtat 1741acatgtatgc aggcaaaaca cccatgcact gacttttaag aaaccctcta gtctgattcc 1801tttcaatttg tcaaatgttg aatgttattt ttaaaatatt ataagccatt taatacaatt 1861tttctttgaa acatggtata gcctagtctg tcttaaattc agaaaaatta tgaagaacaa 1921cattttataa taaagtctta aatgtttcat gtttttg Mouse Aurora kinase B (AURKB)Amino Acid Sequence SEQ. ID. NO. 12MAQKENAYPWPYGSKTSQSGLNTLSQRVLRKEPATTSALALVNRFNSQSTAAPGQKLAENKSQGSTASQGSQNKQPFTIDNFEIGRPLGKGKFGNVYLAREKKSRFIVALKILFKSQIEKEGVEHQLRREIEIQAHLKHPNILQLYNYFYDQQRIYLILEYAPRGELYKELQKSRTFDEQRTATIMEELSDALTYCHKKKVIHRDIKPENLLLGLQGELKIADFGWSVHAPSLRRKTMCGTLDYLPPEMIEGRMHNEMVDLWCIGVLCYELMVGNPPFESPSHSETYRRIVKVDLKFPSSVPSGAQDLISKLLKHNPWQRLPLAEVAAHPWV RANSRRVLPPSALHuman CDK4 mRNA: KR709911 SEQ. ID. NO. 13   1gttcgttgca acaaattgat gagcaatgct tttttataat gccaactttg tacaaaaaag  61ttggcatggc tacctctcga tatgagccag tggctgaaat tggtgtcggt gcctatggga 121cagtgtacaa ggcctgtgat ccccacagtg gccactttgt ggccctcaag agtgtgagag 181tccccaatgg aggaggaggt ggaggaggcc ttcccatcag cacagttcgt gaggtggctt 241tactgaggcg actggaggct tttgagcatc ccaatgttgt ccggctgatg gacgtctgtg 301ccacatcccg aactgaccgg gagatcaagg taaccctggt gtttgagcat gtagaccagg 361acctaaggac atatctggac aaggcacccc caccaggctt gccagccgaa acgatcaagg 421atctgatgcg ccagtttcta agaggcctag atttccttca tgccaattgc atcgttcacc 481gagatctgaa gccagagaac attctggtga caagtggtgg aacagtcaag ctggctgact 541ttggcctggc cagaatctac agctaccaga tggcacttac acccgtggtt gttacactct 601ggtaccgagc tcccgaagtt cttctgcagt ccacatatgc aacacctgtg gacatgtgga 661gtgttggctg tatctttgca gagatgtttc gtcgaaagcc tctcttctgt ggaaactctg 721aagccgacca gttgggcaaa atctttgacc tgattgggct gcctccagag gatgactggc 781ctcgagatgt atccctgccc cgtggagcct ttccccccag agggccccgc ccagtgcagt 841cggtggtacc tgagatggag gagtcgggag cacagctgct gctggaaatg ctgactttta 901acccacacaa gcgaatctct gcctttcgag ctctgcagca ctcttatcta cataaggatg 961aaggtaatcc ggagtgccca actttcttgt acaaagttgg cattataaga aagcattgct 1021tatcaatttg ttgcaacgaa c Human CDK4 Amino Acid Sequence SEQ. ID. NO. 14MATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTVREVALLRRLEAFEHPNVVRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPPPGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE Human CCND1 mRNA: NM_053056 SEQ. ID. NO. 15   1 cacacggact acaggggagt tttgttgaag ttgcaaagtc ctggagcctc cagagggctg  61 tcggcgcagt agcagcgagc agcagagtcc gcacgctccg gcgaggggca gaagagcgcg 121 agggagcgcg gggcagcaga agcgagagcc gagcgcggac ccagccagga cccacagccc 181 tccccagctg cccaggaaga gccccagcca tggaacacca gctcctgtgc tgcgaagtgg 241 aaaccatccg ccgcgcgtac cccgatgcca acctcctcaa cgaccgggtg ctgcgggcca 301 tgctgaaggc ggaggagacc tgcgcgccct cggtgtccta cttcaaatgt gtgcagaagg 361 aggtcctgcc gtccatgcgg aagatcgtcg ccacctggat gctggaggtc tgcgaggaac 421 agaagtgcga ggaggaggtc ttcccgctgg ccatgaacta cctggaccgc ttcctgtcgc 481 tggagcccgt gaaaaagagc cgcctgcagc tgctgggggc cacttgcatg ttcgtggcct 541 ctaagatgaa ggagaccatc cccctgacgg ccgagaagct gtgcatctac accgacaact 601 ccatccggcc cgaggagctg ctgcaaatgg agctgctcct ggtgaacaag ctcaagtgga 661 acctggccgc aatgaccccg cacgatttca ttgaacactt cctctccaaa atgccagagg 721 cggaggagaa caaacagatc atccgcaaac acgcgcagac cttcgttgcc ctctgtgcca 781 cagatgtgaa gttcatttcc aatccgccct ccatggtggc agcggggagc gtggtggccg 841 cagtgcaagg cctgaacctg aggagcccca acaacttcct gtcctactac cgcctcacac 901 gcttcctctc cagagtgatc aagtgtgacc cggactgcct ccgggcctgc caggagcaga 961 tcgaagccct gctggagtca agcctgcgcc aggcccagca gaacatggac cccaaggccg1021 ccgaggagga ggaagaggag gaggaggagg tggacctggc ttgcacaccc accgacgtgc1081 gggacgtgga catctgaggg cgccaggcag gcgggcgcca ccgccacccg cagcgagggc1141 ggagccggcc ccaggtgctc ccctgacagt ccctcctctc cggagcattt tgataccaga1201 agggaaagct tcattctcct tgttgttggt tgttttttcc tttgctcttt cccccttcca1261 tctctgactt aagcaaaaga aaaagattac ccaaaaactg tctttaaaag agagagagag1321 aaaaaaaaaa tagtatttgc ataaccctga gcggtggggg aggagggttg tgctacagat1381 gatagaggat tttatacccc aataatcaac tcgtttttat attaatgtac ttgtttctct1441 gttgtaagaa taggcattaa cacaaaggag gcgtctcggg agaggattag gttccatcct1501 ttacgtgttt aaaaaaaagc ataaaaacat tttaaaaaca tagaaaaatt cagcaaacca1561 tttttaaagt agaagagggt tttaggtaga aaaacatatt cttgtgcttt tcctgataaa1621 gcacagctgt agtggggttc taggcatctc tgtactttgc ttgctcatat gcatgtagtc1681 actttataag tcattgtatg ttattatatt ccgtaggtag atgtgtaacc tcttcacctt1741 attcatggct gaagtcacct cttggttaca gtagcgtagc gtgcccgtgt gcatgtcctt1801 tgcgcctgtg accaccaccc caacaaacca tccagtgaca aaccatccag tggaggtttg1861 tcgggcacca gccagcgtag cagggtcggg aaaggccacc tgtcccactc ctacgatacg1921 ctactataaa gagaagacga aatagtgaca taatatattc tatttttata ctcttcctat1981 ttttgtagtg acctgtttat gagatgctgg ttttctaccc aacggccctg cagccagctc2041 acgtccaggt tcaacccaca gctacttggt ttgtgttctt cttcatattc taaaaccatt2101 ccatttccaa gcactttcag tccaataggt gtaggaaata gcgctgtttt tgttgtgtgt2161 gcagggaggg cagttttcta atggaatggt ttgggaatat ccatgtactt gtttgcaagc2221 aggactttga ggcaagtgtg ggccactgtg gtggcagtgg aggtggggtg tttgggaggc2281 tgcgtgccag tcaagaagaa aaaggtttgc attctcacat tgccaggatg ataagttcct2341 ttccatttct ttaaagaagt tgaagtttag gaatcctttg gtgccaactg gtgtttgaaa2401 gtagggacct cagaggttta cctagagaac aggtggtttt taagggttat cttagatgtt2461 tcacaccgga aggtttttaa acactaaaat atataattta tagttaaggc taaaaagtat2521 atttattgca gaggatgttc ataaggccag tatgatttat aaatgcaatc tccccttgat2581 ttaaacacac agatacacac acacacacac acacacacaa accttctgcc tttgatgtta2641 cagatttaat acagtttatt tttaaagata gatcctttta taggtgagaa aaaaacaatc2701 tggaagaaaa aaaccacaca aagacattga ttcagcctgt ttggcgtttc ccagagtcat2761 ctgattggac aggcatgggt gcaaggaaaa ttagggtact caacctaagt tcggttccga2821 tgaattctta tcccctgccc cttcctttaa aaaacttagt gacaaaatag acaatttgca2881 catcttggct atgtaattct tgtaattttt atttaggaag tgttgaaggg aggtggcaag2941 agtgtggagg ctgacgtgtg agggaggaca ggcgggagga ggtgtgagga ggaggctccc3001 gaggggaagg ggcggtgccc acaccgggga caggccgcag ctccattttc ttattgcgct3061 gctaccgttg acttccaggc acggtttgga aatattcaca tcgcttctgt gtatctcttt3121 cacattgttt gctgctattg gaggatcagt tttttgtttt acaatgtcat atactgccat3181 gtactagttt tagttttctc ttagaacatt gtattacaga tgcctttttt gtagtttttt3241 ttttttttat gtgatcaatt ttgacttaat gtgattactg ctctattcca aaaaggttgc3301 tgtttcacaa tacctcatgc ttcacttagc catggtggac ccagcgggca ggttctgcct3361 gctttggcgg gcagacacgc gggcgcgatc ccacacaggc tggcgggggc cggccccgag3421 gccgcgtgcg tgagaaccgc gccggtgtcc ccagagacca ggctgtgtcc ctcttctctt3481 ccctgcgcct gtgatgctgg gcacttcatc tgatcggggg cgtagcatca tagtagtttt3541 tacagctgtg ttattctttg cgtgtagcta tggaagttgc ataattatta ttattattat3601 tataacaagt gtgtcttacg tgccaccacg gcgttgtacc tgtaggactc tcattcggga3661 tgattggaat agcttctgga atttgttcaa gttttgggta tgtttaatct gttatgtact3721 agtgttctgt ttgttattgt tttgttaatt acaccataat gctaatttaa agagactcca3781 aatctcaatg aagccagctc acagtgctgt gtgccccggt cacctagcaa gctgccgaac3841 caaaagaatt tgcaccccgc tgcgggccca cgtggttggg gccctgccct ggcagggtca3901 tcctgtgctc ggaggccatc tcgggcacag gcccaccccg ccccacccct ccagaacacg3961 gctcacgctt acctcaacca tcctggctgc ggcgtctgtc tgaaccacgc gggggccttg4021 agggacgctt tgtctgtcgt gatggggcaa gggcacaagt cctggatgtt gtgtgtatcg4081 agaggccaaa ggctggtggc aagtgcacgg ggcacagcgg agtctgtcct gtgacgcgca4141 agtctgaggg tctgggcggc gggcggctgg gtctgtgcat ttctggttgc accgcggcgc4201 ttcccagcac caacatgtaa ccggcatgtt tccagcagaa gacaaaaaga caaacatgaa4261 agtctagaaa taaaactggt aaaaccccaa aaaaaaaaaa aaaa Human CCND1Amino Acid Sequence SEQ. ID. NO. 16MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI Mouse CDK4 mRNA: NM_009870 SEQ. ID. NO. 17    1gtgggggtga gggggcctct ctagctcgcg gcctgtgtct atggtctggc ccgaagcgtc   61cagctgcccg ggaccgatcc ccggtgtatg gcgccgcagg aaccggctcc cgggcccaga  121taaagggcca cctccagagc tcttagccga gcgtaagatc ccctgcttcg agaatggctg  181ccactcgata tgaacccgtg gctgaaattg gtgtcggtgc ctatgggacg gtgtacaaag  241cccgagatcc ccacagtggc cactttgtgg ccctcaagag tgtgagagtt cctaatggag  301gagcagctgg agggggcctt cccgtcagca cagttcgtga ggtggccttg ttaaggaggc  361tggaggcctt tgaacatccc aatgttgtac ggctgatgga tgtctgtgct acttcccgaa  421ctgatcggga catcaaggtc accctagtgt ttgagcatat agaccaggac ctgaggacat  481acctggacaa agcacctcca ccgggcctgc cggttgagac cattaaggat ctaatgcgtc  541agtttctaag cggcctggat tttcttcatg caaactgcat tgttcaccgg gacctgaagc  601cagagaacat tctagtgaca agtaatggga ccgtcaagct ggctgacttt ggcctagcta  661gaatctacag ctaccagatg gccctcacgc ctgtggtggt tacgctctgg taccgagctc  721ctgaagttct tctgcagtct acatacgcaa cacccgtgga catgtggagc gttggctgta  781tctttgcaga gatgttccgt cggaagcctc tcttctgtgg aaactctgaa gccgaccagt  841tggggaaaat ctttgatctc attggattgc ctccagaaga cgactggcct cgagaggtat  901ctctacctcg aggagccttt gcccccagag ggcctcggcc agtgcagtca gtggtgccag  961agatggagga gtctggagcg cagctgctac tggaaatgct gacctttaac ccacataagc 1021gaatctctgc cttccgagcc ctgcagcact cctacctgca caaggaggaa agcgacgcag 1081agtgagaaga ggggctgcct ttcccagtct tggtggagaa accctcgctg aagcggcagc 1141ctctgtttcc ccccaaggct gtggagaatc ctccagtttt ttacagagaa tattttaagc 1201cttaaataac aagtccccac ctctccttac gaggttcacc cccattaccc tcccctagct 1261ctacactaaa gggcaggtgt atctgtcttc ttccctccct gatttatact gggatctttt 1321ttatacagga aaacaagaca agacaaaaaa aaaaaaaaaa aaaaa Mouse CDK4Amino Acid Sequence SEQ. ID. NO. 18MAATRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGAAGGGLPVSTVREVALLRRLEAFEHPNVVRLMDVCATSRTDRDIKVTLVFEHIDQDLRTYLDKAPPPGLPVETIKDLMRQFLSGLDFLHANCIVHRDLKPENILVTSNGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPREVSLPRGAFAPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKEESDAE Mouse CCDN1 mRNA: NM_007631.2SEQ. ID. NO. 19    1ttttctctgc ccggctttga tctctgctta acaacagtaa cgtcacacgg actacagggg   61agttttgttg aagttgcaaa gtcctgcagc ctccagaggg ctgtcggcgc agtagcagag  121agctacagac tccgcgcgct ccggagaccg gcagtacagc gcgaggcagc gcgcgtcagc  181agccgccacc ggagcccaac cgagaccaca gccctcccca gacggccgcg ccatggaaca  241ccagctcctg tgctgcgaag tggagaccat ccgccgcgcg taccctgaca ccaatctcct  301caacgaccgg gtgctgcgag ccatgctcaa gacggaggag acctgtgcgc cctccgtatc  361ttacttcaag tgcgtgcaga aggagattgt gccatccatg cggaaaatcg tggccacctg  421gatgctggag gtctgtgagg agcagaagtg cgaagaggag gtcttcccgc tggccatgaa  481ctacctggac cgcttcctgt ccctggagcc cttgaagaag agccgcctgc agctgctggg  541ggccacctgc atgttcgtgg cctctaagat gaaggagacc attcccttga ctgccgagaa  601gttgtgcatc tacactgaca actctatccg gcccgaggag ctgctgcaaa tggaactgct  661tctggtgaac aagctcaagt ggaacctggc cgccatgact ccccacgatt tcatcgaaca  721cttcctctcc aaaatgccag aggcggatga gaacaagcag accatccgca agcatgcaca  781gacctttgtg gccctctgtg ccacagatgt gaagttcatt tccaacccac cctccatggt  841agctgctggg agcgtggtgg ctgcgatgca aggcctgaac ctgggcagcc ccaacaactt  901cctctcctgc taccgcacaa cgcactttct ttccagagtc atcaagtgtg acccggactg  961cctccgtgcc tgccaggaac agattgaagc ccttctggag tcaagcctgc gccaggccca 1021gcagaacgtc gaccccaagg ccactgagga ggagggggaa gtggaggaag aggctggtct 1081ggcctgcacg cccaccgacg tgcgagatgt ggacatctga gggccaccgg gcaggcggga 1141gccaccaagt agtggcaccc gcaaagagga aggagccagc ccgggtgctc ctgacgacgt 1201cccccttggg gacatgttgt taccagaaga ggaagttttg ttctctttgt tggttgtttt 1261tccttaatct ttctcctttc tatctgattt aagcaaaaga gaaaaaaata tctgaaagct 1321gtcttaaaga gagagagaga gagatagaat ctgcatcacc ctgagagtag ggagccaggg 1381ggtgctacaa aaatagaatt ctgtacccca gtaatcaact agttttctat taatgtgctt 1441gtctgttcta agagtaggat taacacaggg gaagtcttga gaaggagttt tgattctttt 1501atatgttttt aaaaaaaagc ttaagaaaca ttgctttaaa aaggaaggaa aaaaaataca 1561gcaaaccatt gttaaagtag aagagttttt aggttgagaa atgtactctg ctttgctgaa 1621aagccacagc ttaggccctc agcctcactc cctggcttgc tcagtgccta cagccctgtt 1681acctgatacc tgtgctttat cccaggggtg ggcagacctc ttaaccttat agatggtcag 1741tgcgacctct agtggtctca tggcgtgtgg cacaaccccc ctccccaggg ctcagcttaa 1801tgtgccctct ccccccaaca acctgcaggt tcacagcacc agccacacag cggtagggat 1861gaaatagtga cataatatat tctatttttg taaccttcct attttgtagc tctgtttaga 1921gagatgctgg tttttgcctg aaggccctgc agcctgccca catcaggtta aacccacagc 1981ttttgtgtgt ggtttgtttt gttgtgtttt ctttctctat gttccaaaac cattccattt 2041caaagcactt ttggtcagct agctggaggc agtgttgctg gtgtgtgttg gggggagggg 2101ttctaatgga atggatgggg atgtccacac acgcattcag atggctgtac aacaggttgt 2161agggctggta gtatgaggtg cttgggaagt tttgttgggt caagaagaga gaactctgtt 2221ctcgcaccac cgggatctgt cctgcaaagt tgaagggatc ctttggtgcc agctggtgtt 2281tggaagtagg aaccatgatg gcattacctg gacaaggaga ttggggacaa ctcttaagtc 2341tcacacagga ggcttttaaa cactaaaatg tctaatttat acttaaggct acagaagagt 2401atttatggga aaggctgccc atgaccagtg tgactcaaag caatgtgatc tcccttgatt 2461caaacgcaca cctctgccct gctggagaag gtttagggcc atgtctgaga gattggtctt 2521tcattgggca acgggggggg ggggggggtc cttaaaaaaa aaaaaccaca aagacagaga 2581tttggtctgc ttgactttcc caacccaatt ggccccattg gagagccatc caaactgagg 2641aaaattaggg gactccaaaa gagtttgatt ctggcacatt cttgccgctg cccccaagtt 2701aacaacagta ggtaatttgc acacctctgg ctctgtgcct ttctattagg actttttggc 2761agaaggtgga gagcgggagg cttaagaggg gatgtgaggg aagaggtgaa ggtgggacca 2821catgggacag gccacggctc ctctcatggc gctgctaccg atgactccca ggatcccaga 2881cgttcagaac cagattctca ttgctttgta tctttcacgt tgttttcgct gctattggag 2941ggtcagtttt gattgttttt gttttacaat gtcagactgc catgttcaag ttttaatttc 3001ctcatagagt gtatttacag atgccctttt ttgtactttt ttttttaatt gtgatctatt 3061ttggcttaat gtgattaccg ctgtattcca aaaaaaaaaa aaaaacaggt tcctgttcac 3121aatacctcat gtatcatcta gccatgcacg agcctggcag gcaggtgggc ggtctgcctc 3181cagggatcct gggaccctga tggcgatcgt cctgtcatgc tgggcccttc atttgatctg 3241ggacatagca tcacagcagt cagggcacct ggattgttct gttatcgata ttgtttcttg 3301tagcggcctg ttgtgcatgc caccatgctg ctggcccggg gggatttgct ctgagtctcc 3361ggtgcatcat ttaatctgtt aggttctagt gttccgtctt gttttgtgtt aattacagca 3421ttgtgctaat gtaaagactc tgcctttgcg aagccagctg cagtgctgta ggcccccaag 3481ttccctagca agctgccaaa ccaaaacggg caccaccagc tcagctgagg catcccagcc 3541aggcaggacc cttgagggcc gctgtatcca tggtgatggg gtgaggtttt ggccaaaagg 3601ccaaagactg gtggtgggtc cacggaatct gccctgtgac atgaaaggct ttgaggggct 3661ctggctggtg gccaggttgg ctttttgtat ttctggttga cacaccatgg cgcttcccag 3721cacagacatg tgaccagcat ggtccaggaa aaaaaaaaag acaaaaaatc tagaaaataa 3781aattggtaaa atctca Mouse CCND1 Amino Acid Sequence SEQ. ID. NO. 20MEHQLLCCEVETIRRAYPDTNLLNDRVLRAMLKTEETCAPSVSYFKCVQKEIVPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPLKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEADENKQTIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAMQGLNLGSPNNFLSCYRTTHFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNVDPKATEEEGEVEEEAGLACTPTDVRDVDI

What is claimed is:
 1. A method of inducing proliferation and/or cellcycle reentry of an adult postmitotic cell, the method comprisingcontacting the adult postmitotic cell with an effective amount of acomposition comprising cyclin-dependent kinase-1 (CDK1), cyclinB, CDK4,and cyclinD, or equivalents of each thereof, thereby inducingproliferation and/or cell cycle reentry of the adult postmitotic cell.2. The method of claim 1, further comprising contacting the cell with aneffective amount of a CDK activator, a transforming growth factor βinhibitor, or combinations thereof.
 3. The method of claim 2, whereinthe CDK activator is a CDK1 activator.
 4. The method of claim 1, whereinthe adult postmitotic cell is selected from the group consisting of acardiomyocyte, a neural cell, a pancreatic cell, a hair cell, and askeletal muscle cell.
 5. The method of claim 1, wherein thecyclin-dependent kinase-1 (CDK1), cyclinB, CDK4, and cyclinD encoded byat least one heterologous nucleic acid.
 6. The method of claim 5,wherein the at least one nucleic acid is a modified mRNA.
 7. The methodof claim 6, wherein the at least one nucleic acid is constitutivelyexpressed.
 8. The method of claim 1, wherein the cyclinB is cyclinB1(CCNB1), and/or the cyclinD is cyclinD1 (CCND1).
 9. The method of claim1, wherein the CDK1 and/or CDK4 is constitutively expressed.